Oxidative damage in age-related macular degeneration.

Epidemiologic studies have suggested that elderly patients who consumed diets rich in antioxidants throughout their lives are less likely to be afflicted with age-related macular degeneration (AMD). This led to the Age-Related Eye Disease Study, which showed that supplements containing antioxidant vitamins and zinc reduce the risk of progression to severe stages of AMD. Despite these data that indirectly implicate oxidative damage in the pathogenesis of AMD, there has not been any direct demonstration of increased oxidative damage in the retinas of patients with AMD. In this study, we used biomarkers of oxidative damage in postmortem eyes from patients with AMD and comparably aged patients without AMD to directly assess for oxidative damage. Sections from 4 eyes with no pathologic features of AMD showed no immunofluorescent staining for markers of oxidative damage, while sections from 8 of 12 eyes with advanced geographic atrophy showed evidence of widespread oxidative damage in both posterior and anterior retina. Only 2 of 8 eyes with choroidal neovascularization and 2 of 16 eyes with diffuse drusen and no other signs of AMD showed evidence of oxidative damage. These data suggest that widespread oxidative damage occurs in the retina of some patients with AMD and is more likely to be seen in patients with advanced geographic atrophy. This does not rule out oxidative damage as a pathogenic mechanism in patients with CNV, but suggests that a subpopulation of patients with geographic atrophy may have a major deficiency in the oxidative defense system that puts the majority of cells in the retina at risk for oxidative damage.

H-Ras oncogene counteracts the growth-inhibitory effect of genistein in T24 bladder carcinoma cells.

Among eight human bladder cancer cell lines we examined, only T24 cells were resistant to the growth inhibition effect of genistein, an isoflavone and potent anticancer drug. Since the T24 cell line was the only cell line known to overexpress oncogenic H-Ras(val 12), we investigated the role of H-Ras(val 12) in mediating drug resistance. Herein, we demonstrate that the phenotype of T24 cells could be dramatically reversed and became relatively susceptible to growth inhibition by genistein if the synthesis of H-Ras(val 12) or its downstream effector c-Fos had been suppressed. The inhibition of Ras-mediated signalling with protein kinase inhibitors, such as PD58059 and U0126 which inhibited MEK and ERK, in T24 cells also rendered the identical phenotypic reversion. However, this reversion was not observed when an inhibitor was used to suppress the protein phosphorylation function of PI3 K or PKC. These results suggest that the signal mediated by H-Ras(val 12) is predominantly responsible for the resistance of the cells to the anticancer drug genistein.

Potential clozapine target sites on peripheral hematopoietic cells and stromal cells of the bone marrow.

The antipsychotic drug clozapine, acts via interaction with selective neurotransmitter receptor systems. Its use however, is associated with life-threatening agranulocytosis. The mechanism by which this occurs and its possible relationship with the drug's atypicality remain unclear. As a first step in identifying mechanistic pathways involved, profiling of neurotransmitter receptors on human neutrophils, mononuclear and bone marrow stromal cells as putative targets for clozapine-mediated toxicity was undertaken. Expression of mRNA encoding dopaminergic d2, d3, d4; serotonergic 5ht2a, 5ht2c, 5ht3, 5ht6, 5ht7; adrenergic alpha1a, alpha2; histaminergic h1 and muscarinic m1, m2, m3, m4, m5 receptors was analyzed by reverse transcription-polymerase chain reaction methods. While 5ht2c, 5ht6, m1 and m2 mRNA were undetected, the presence of the other receptors indicates sites at which clozapine could bind and induce toxicity of neutrophils and stromal components which regulate granulopoiesis. The functional significance of differential receptor expression while unknown, may argue for neural regulation of hematopoiesis.

Role of splenectomy in immune (idiopathic) thrombocytopenic purpura.

Idiopathic thrombocytopenic purpura (ITP) is an immune disorder, which causes an acute or chronic thrombocytopenia, and may result in potentially life-threatening hemorrhage. Splenectomy is one of the treatment options that needs to be weighed in the treatment of ITP, particularly in cases that have shown response failure to medical modalities such as prednisone, i.v.Ig, or anti-D globulin therapy. Although most studies demonstrate good early response following splenectomy, the long-term outcome is less favorable. Furthermore, other negative factors, such as rendering the patient ineligible for anti-D globulin or oral tolerance therapy and vulnerable to possible life-threatening sepsis, must be weighed prior to splenectomy.

Megakaryocyte-targeted synthesis of the integrin beta(3)-subunit results in the phenotypic correction of Glanzmann thrombasthenia.

Glanzmann thrombasthenia is an inherited bleeding disorder characterized by qualitative or quantitative defects of the platelet-specific integrin, alphaIIbbeta(3). As a result, alphaIIbbeta(3) cannot be activated and cannot bind to fibrinogen, leading to a loss of platelet aggregation. Thrombasthenia is clinically characterized by mucocutaneous hemorrhage with episodes of intracranial and gastrointestinal bleeding. To develop methods for gene therapy of Glanzmann thrombasthenia, a murine leukemia virus (MuLV)-derived vector, -889Pl(A2)beta(3), was transduced into peripheral blood CD34(+) cells from 2 patients with thrombasthenia with defects in the beta(3) gene. The human alphaIIb promoter was used in this vector to drive megakaryocyte-targeted expression of the wild-type beta(3) subunit. Proviral DNA and alphaIIbbeta(3) biosynthesis were detected after in vitro differentiation of transduced thrombasthenic CD34(+) cells with megakaryocyte growth and development factor. Flow cytometric analysis of transduced patient samples indicated that 19% of megakaryocyte progeny expressed alphaIIbbeta(3) on the surface at 34% of normal receptor levels. Treatment of transduced megakaryocytes with a combination of agonists including epinephrine and the thrombin receptor-activating peptide induced the alphaIIbbeta(3) complex to form an activated conformation capable of binding fibrinogen as measured by PAC-1 antibody binding. Transduced cells retracted a fibrin clot in vitro similar to megakaryocytes derived from a normal nonthrombasthenic individual. These results demonstrate ex vivo phenotypic correction of Glanzmann thrombasthenia and support the potential use of hematopoietic CD34(+) cells as targets for alphaIIb promoter-driven MuLV vectors for gene therapy of platelet disorders. (Blood. 2000;95:3645-3651)

Thrombotic thrombocytopenic purpura associated with clopidogrel.

BACKGROUND: The antiplatelet drug clopidogrel is a new thienopyridine derivative whose mechanism of action and chemical structure are similar to those of ticlopidine. The estimated incidence of ticlopidine-associated thrombotic thrombocytopenic purpura is 1 per 1600 to 5000 patients treated, whereas no clopidogrel-associated cases were observed among 20,000 closely monitored patients treated in phase 3 clinical trials and cohort studies. Because of the association between ticlopidine use and thrombotic thrombocytopenic purpura and other adverse effects, clopidogrel has largely replaced ticlopidine in clinical practice. More than 3 million patients have received clopidogrel. We report the clinical and laboratory findings in 11 patients in whom thrombotic thrombocytopenic purpura developed during or soon after treatment with clopidogrel. METHODS: The 11 patients were identified by active surveillance by the medical directors of blood banks (3 patients), hematologists (6), and the manufacturer of clopidogrel (2). RESULTS: Ten of the 11 patients received clopidogrel for 14 days or less before the onset of thrombotic thrombocytopenic purpura. Although 10 of the 11 patients had a response to plasma exchange, 2 required 20 or more exchanges before clinical improvement occurred, and 2 had relapses while not receiving clopidogrel. One patient died despite undergoing plasma exchange soon after diagnosis. CONCLUSIONS: Thrombotic thrombocytopenic purpura can occur after the initiation of clopidogrel therapy, often within the first two weeks of treatment. Physicians should be aware of the possibility of this syndrome when initiating clopidogrel treatment.

Long-term outcome of splenectomy for idiopathic thrombocytopenic purpura.

Idiopathic thrombocytopenic purpura (ITP) is an illness of primary acquired thrombocytopenia occurring in the absence of marrow failure. Splenectomy was first used as a treatment for ITP in 1913. However, with the realization that opsonin (critical for the optimal killing of invasive micro-organisms by white blood cells) is manufactured only in the spleen, spontaneous splenic removal was reevaluated and questioned. Splenectomy has a success rate that remains nearly identical (about 50% to 60%) whether it is performed soon after diagnosis or several months or years later. As yet, there is no consistently effective method to predict an individual ITP patient's response to splenectomy. As the time since splenectomy increases, however, the rate of excellent response decreases. Despite pneumococcal vaccination prior to splenectomy, fatal fulminant sepsis is an omnipresent possibility. Because a number of published studies, including the Johns Hopkins experience, have questioned the long-term outcome of splenectomy, splenectomy should not be the first treatment option for ITP patients. It should be performed only after all other therapeutic modalities have been exhausted, and the patient has a platelet count less than 25,000/microL and is hemorrhaging. Once patients have undergone splenectomy, they are Ineligible for potentially excellent treatment such as anti-D globulin or oral tolerance therapy.

Persistant pre-eclampsia post partum with elevated liver enzymes and hemolytic uremic syndrome.

The spectrum of complications with pre-eclampsia, which may include AFLP (acute fatty liver of pregnancy) as well as the HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets), is resolved by early delivery. However, the ravages of HUS/TTP (hemolytic uremic syndrome/thrombotic thrombocytopenic purpura) require therapy usually by plasma exchange. Overlap between these two groups of syndromes has occurred on rare occasions and usually requires the therapy of the predominant or more dangerous or threatening form. Such overlap can be appreciated and then treated successfully without residual morbidity. The index case is presented and an extensive review of the two groups of syndromes is provided.

Oral contraceptives and smoking, current considerations: recommendations of a consensus panel.

In a closed meeting, members of the consensus panel evaluated the presentations of the scientific panel and developed a series of recommendations. They outlined clinical imperatives related to the identification and education of patients who smoke, the physician's role in smoking cessation, and the prescription of oral contraceptives for patients who smoke. They also outlined research objectives for the future. The most important suggestions include the following: All patients should be asked about their smoking status at every visit, and all smokers should be encouraged and helped to quit. The decision to prescribe an oral contraceptive requires a detailed personal and family history of thrombotic disease. Measurement of lipid profile should be considered, along with exercise and dietary intervention, for smokers >35 years old who use or request oral contraceptives. Patients >35 years old who smoke heavily (>15 cigarettes/d) should be denied the use of oral contraceptives. Preliminary data suggest that an oral contraceptive with the very low dose of 20 micrograms ethinyl estradiol may be safer for oral contraceptive users who smoke, even for those >35 years old who have an occasional cigarette, but these laboratory findings require clinical corroboration.

PIP: This article summarizes the activities of a conference in Montreal, Canada, entitled "Oral Contraceptives and Smoking: Current Considerations." Members of the consensus panel evaluated the presentations of the scientific panel and developed a series of recommendations. The clinical imperatives related to the identification and education of patients who smoke, physicians¿ role in smoking cessation, and prescription of OCs for patients who smoke were elaborated. The following recommendations were made: 1) encourage patients who smoke to quit smoking; 2) counsel patients regarding OC benefits; furthermore, counsel patients concerning the risks of concomitant smoking and OC use; and 3) prescribe 20 mcg ethinyl estradiol for women who smoke. In view of the remaining unanswered questions, the panel outlined several research objectives for the future.

Clozapine treatment in Australia: a review of haematological monitoring.

BACKGROUND: Clozapine is an atypical antipsychotic drug indicated for patients with schizophrenia in whom traditional antipsychotic drugs (such as haloperidol or the phenothiazines) are ineffective, or in those who experience intolerable adverse effects. Clozapine treatment may be complicated by the development of life-threatening agranulocytosis, so regular haematological monitoring is required. OBJECTIVES: To determine the incidence of clozapine-induced agranulocytosis in Australia and the importance of monitoring white blood cell counts in patients treated with clozapine. DESIGN: Review of haematological monitoring for the first three years (June 1993-July 1996) of operation of the Australian Clozaril (clozapine; Novartis Australia) Patient Monitoring System (CPMS) central database. RESULTS: In the 4061 patients prospectively monitored by the CPMS, the incidence of agranulocytosis, neutropenia and leukopenia combined was 2.6% (n = 104); the incidence of agranulocytosis was 0.9% (n = 37). So far there have been no deaths in Australia from the complications of clozapine-induced agranulocytosis. CONCLUSION: The incidence of agranulocytosis and neutropenia associated with clozapine use in Australia is similar to that in the rest of the world. Monitoring the white blood cell counts of patients being treated with clozapine ensures minimal risk to patients who develop agranulocytosis.

Thrombocytopenia in pregnancy.

Thrombocytopenia is a common finding in normal pregnancy. The introduction of routine automated complete blood counting has clearly documented this. In the past, these patients would go undetected and be spared unnecessary testing, procedures, or medications. This article reviews the common causes of thrombocytopenia and offers criteria on which the diagnosis of clinically significant thrombocytopenia can be made. In addition, we will discuss therapeutic approaches to manage patients with pathologic thrombocytopenia.

Evaluation of thrombolytic agents.

The mechanism of action of currently available thrombolytic agents, such as streptokinase, urokinase, alteplase (recombinant tissue-type plasminogen activator; rt-PA) and anistreplase (anisoylated plasminogen streptokinase activator complex; APSAC), involves conversion of inactive plasminogen to plasmin, a potent fibrinolytic. However, the relatively weak substrate specificity of first generation agents (streptokinase and urokinase) can result in a state of systemic fibrinolysis and associated bleeding complications. The second generation drugs such as alteplase were developed in an attempt to enhance fibrin specificity, so that only enzymatic conversion of fibrin-complexed plasminogen would take place, thus avoiding systemic fibrinolysis. Results from large clinical trials have failed to consistently show any significant differences between first and second generation thrombolytic agents in the incidence of bleeding. In the clinical setting, thrombolytic agents have been evaluated primarily in patients with acute myocardial infarction and have been shown to significantly reduce morbidity and mortality compared with conservative treatment. The focus of current and future research is to investigate these agents in patients with other vaso-occlusive or ischaemic conditions (e.g. stroke), and also to evaluate different drug administration regimens and the use of adjunctive therapies such as aspirin (acetylsalicylic acid) and heparin.

Defibrinogenating enzymes.

The venoms from 3 snakes have been shown to induce defibrinogenation: ancrod from the venom of Calloselasma rhodostoma (formerly known as Agkistrodon rhodostoma), batroxobin from the venom of Bothrops atrox moojeni, and crotalase from the venom of Crotalus adamanteus. The purified fractions of ancrod, batroxobin, and crotalase possess coagulant, proteolytic and esterolytic properties, although their primary mechanism of action is a proteolytic effect on circulating fibrinogen. Ancrod cleaves only the A-fibrinopeptides, but not the B-fibrinopeptides, from fibrinogen; this contrasts with thrombin, batroxobin and crotalase, which cleave both fibrinopeptides A and B. Within minutes of administration of ancrod or batroxobin, there is a significant reduction in plasma fibrinogen levels, and these remain exceedingly low with repeated administration (once or twice daily). The rapid fall in plasma fibrinogen levels is accompanied by a slightly delayed but marked rise in the level of fibrinogen-fibrin degradation products. Plasminogen levels are decreased and blood viscosity is reduced, but formed elements in the circulating blood remain unaltered. Ancrod and batroxobin have been investigated in patients with stroke, deep-vein thrombosis, myocardial infarction, peripheral arterial thrombosis, priapism, and sickle-cell crisis; crotalase has not been administered to humans. However, results have been difficult to interpret, and additional well designed trials are needed to better define the optimum role of ancrod and batroxobin in the management of these conditions. Overall, treatment is well tolerated and serious adverse events are infrequent. In the coagulation laboratory, ancrod, batroxobin and crotalase may be used as reagents to perform coagulation studies on specimens of blood that contain heparin. These venom fractions can be substituted for thrombin in performing the thrombin time and in removing fibrinogen from plasma for accurate determination of fibrinogen-fibrin degradation products.

Glanzmann thrombasthenia. Cooperation between sequence variants in cis during splice site selection.

Glanzmann thrombasthenia (GT), an autosomal recessive bleeding disorder, results from abnormalities in the platelet fibrinogen receptor, GP(IIb)-IIIa (integrin alpha(IIb)beta3). A patient with GT was identified as homozygous for a G-->A mutation 6 bp upstream of the GP(IIIa) exon 9 splice donor site. Patient platelet GP(IIIa) transcripts lacked exon 9 despite normal DNA sequence in all of the cis-acting sequences known to regulate splice site selection. In vitro analysis of transcripts generated from mini-gene constructs demonstrated that exon skipping occurred only when the G-->A mutation was cis to a polymorphism 116 bp upstream, providing precedence that two sequence variations in the same exon which do not alter consensus splice sites and do not generate missense or nonsense mutations, can affect splice site selection. The mutant transcript resulted from utilization of a cryptic splice acceptor site and returned the open reading frame. These data support the hypothesis that pre-mRNA secondary structure and allelic sequence variants can influence splicing and provide new insight into the regulated control of RNA processing. In addition, haplotype analysis suggested that the patient has two identical copies of chromosome 17. Markers studied on three other chromosomes suggested this finding was not due to consanguinity. The restricted phenotype in this patient may provide information regarding the expression of potentially imprinted genes on chromosome 17.

The fibrinolytic system in neoplasia.

During activation of the fibrinolytic system plasminogen is converted to plasmin by tissue plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). t-PA is predominantly released from endothelial cells, u-PA primarily by renal parenchymal cells. The activation of plasminogen is regulated by plasminogen activator inhibitor-1 (PAI-1), plasmin is controlled by alpha 2-plasmin inhibitor. The fibrinolytic system is not only involved in the intravascular dissolution of fibrin (thrombi), it also plays a vital role in normal physiologic reproduction, wound repair, angiogenesis, and tissue remodeling. Fibrinolysis is also a vital component in the pathogenesis of neoplastic disease. It is essential in releasing cells from their primary site of origin, providing nutrition for neoplastic cell growth and promoting cell mobility and motility. In neoplastic cells the degradation of the extracellular matrix proteins is facilitated by excessive expression of u-PA, t-PA, and u-PAR. In many forms of carcinoma increased expression of u-PAR and u-PA is associated with significantly shorter survival. Greater expression of u-PA in breast cancer cells, for example, is associated with shorter survival and increased relapse rate. Progressively aggressive neoplastic cells evidence high expression of u-PA and u-PAR activities, variable expression of t-PA, and enhanced PAI-1 and PAI-2 activities. In acute nonlymphocytic leukemias, poor outcome correlates with high t-PA levels. In acute progranulocytic leukemia there is a high incidence of DIC. Neoplastic prostatic tissue also expresses high u-PA activity and the more aggressive the cell line, the greater the number of u-PAR and the higher the u-PA activity. In gynecologic malignancies, a greater expression of u-PA in combination with cathepsin D is associated with widespread disease and poor prognosis. High u-PA values were also seen in patients with brain, gastric, and hepatic malignancies. It is evident that the plasminogen-plasmin system is a vital component in the biology of neoplastic disease and that it is, in theses conditions, in no way beneficial to the host.

Laboratory monitoring of thrombolytic therapy.

The concept and strategy of thrombolytic therapy are absolutely correct. The obstructing intraluminal thrombus is in urgent need of resolution to permit reestablishment of blood flow. As investigative efforts in this area continue, more effective and more rapidly acting thrombolytic agents will be forthcoming. The thrombolytic agents available today are excellent and can definitely be used safely. However, a modicum of common sense must be employed in initiating their use. There must be frequent contact with the patient receiving such important and needed therapy. It is foolish for those individuals either not familiar with or not educated to utilize thrombolytic agents to make broad sweeping statements that such agents, whose efficacy has been demonstrated, should not be employed in the treatment of thrombosis. Such an attitude is an example of how not to proceed to further improvement in health care. To be content to remain in the past will never permit entrance into the future.