Increased risk of non-Q wave myocardial infarction after directional atherectomy is platelet dependent: evidence from the EPIC trial. Evaluation of c7E3 for the Prevention of Ischemic Complications.

OBJECTIVES: We sought to determine the effects of platelet glycoprotein IIb/IIIa receptor blockade on adverse outcomes, especially non-Q wave myocardial infarction, in patients undergoing directional atherectomy in the Evaluation of c7E3 for the Prevention of Ischemic Complications (EPIC) trial. BACKGROUND: Randomized trials comparing directional atherectomy with percutaneous transluminal coronary angioplasty (PTCA) have demonstrated modest benefits favoring atherectomy but at a cost of increased acute ischemic complications, notably non-Q wave myocardial infarction. The mechanism for this excess risk is unknown. METHODS: Of 2,038 high risk patients undergoing coronary intervention in the EPIC trial, directional atherectomy was performed in 197 (10%). Patients randomly received the chimeric glycoprotein IIb/IIIa antibody 7E3 (c7E3), as a bolus or a bolus and 12-h infusion or placebo. Study end points included death, myocardial infarction, repeat intervention or bypass surgery. RESULTS: Patients undergoing directional atherectomy had a lower baseline risk for acute complications but had a higher incidence of any myocardial infarction (10.7% vs. 6.3%, p = 0.021) and non-Q wave myocardial infarction (9.6% vs. 4.9%, p = 0.006). Bolus and infusion of c7E3 reduced non-Q wave myocardial infarctions by 71% after atherectomy (15.4% for placebo vs. 4.5% for bolus and infusion, p = 0.046). Non-Q wave myocardial infarction rates after PTCA were not affected by c7E3, although Q wave myocardial infarctions were reduced from 2.6% to 0.8% (p = 0.017). CONCLUSIONS: The EPIC trial confirmed the increased risk of non-Q wave myocardial infarction with directional atherectomy use compared with PTCA. A bolus and 12-h infusion of the glycoprotein IIb/IIIa receptor inhibitor c7E3 abolished this excess risk. Directional atherectomy-related non-Q wave myocardial infarction appears to be platelet aggregation dependent.

Polyomavirus models of brain infection and the pathogenesis of multiple sclerosis.

Multiple sclerosis (MS) is generally considered to be an autoimmune disorder with myelin as the target and with several unidentified viruses playing ancillary roles, possibly through molecular mimicry. Although this paradigm has led to important progress on potential mechanisms of myelin loss, neither a target antigen in myelin nor a triggering mechanism has yet been identified, leaving the etiology of MS still unknown. Animal models of viral demyelination and studies showing that JC virus (JCV), the polyomavirus which causes progressive multifocal leukoencephalopathy (PML), may be latent in some normal human brains suggest another possibility. A host immune response targeting proteins expressed at low levels from viral DNA latent in the central nervous system (CNS) might underlie a focal demyelinating disease such as MS. A shift from autoimmunity to a latent-virus model is not a trivial substitution of target antigens. This shift would expand the search for a definitive laboratory test for MS and could lead to improved therapeutic and preventive approaches.

JC virus as a marker of human migration to the Americas.

JC virus is a ubiquitous human polyomavirus present in populations worldwide. Seven genotypes differing in DNA sequence by approximately 1-3% characterize three Old World population groups (African, European and Asian) as well as Oceania. It is possible to follow Old World populations into the New World by the JC virus genotypes they carried. The first population to settle in the Americas, the Native Americans, brought with them type 2A from northeast Asia. European settlers arriving after Columbus carried primarily type 1 and type 4. Africans brought by the slave trade carried type 3 and type 6.

Human polyomavirus JC variants in Papua New Guinea and Guam reflect ancient population settlement and viral evolution.

The peopling of the Pacific was a complex sequence of events that is best reconstructed by reconciling insights from various disciplines. Here we analyze the human polyomavirus JC (JCV) in Highlanders of Papua New Guinea (PNG), in Austronesian-speaking Tolai people on the island of New Britain, and in nearby non-Austronesian-speaking Baining people. We also characterize JCV from the Chamorro of Guam, a Micronesian population. All JCV strains from PNG and Guam fall within the broad Asian group previously defined in the VP1 gene as Type 2 or Type 7, but the PNG strains were distinct from both genotypes. Among the Chamorro JCV samples, 8 strains (Guam-1) were like the Type 7 strains found in Southeast Asia, while nine strains (Guam-2) were distinct from both the mainland strains and most PNG strains. We identified three JCV variants within Papua New Guinea (PNG-1, PNG-2 and PNG-3), but none of the Southeast Asian (Type 7) strains. PNG-1 strains were present in all three populations (Highlanders and the Baining and Tolai of New Britain), but PNG-2 strains were restricted to the Highlanders. Their relative lack of DNA sequence variation suggests that they arose comparatively recently. The single PNG-3 strain, identified in an Austronesian-speaking Tolai individual, was closely related to the Chamorro variants (Guam-2), consistent with a common Austronesian ancestor. In PNG-2 variants a complex regulatory region mutation inserts a duplication into a nearby deletion, a change reminiscent of those seen in the brains of progressive multifocal leukoencephalopathy patients. This is the first instance of a complex JCV rearrangement circulating in a human population.

A monoclonal antibody to SV40 large T-antigen labels a nuclear antigen in JC virus-transformed cells and in progressive multifocal leukoencephalopathy (PML) brain infected with JC virus.

Thirty monoclonal antibodies to SV40 large T-antigen were tested for reactivity on the JC virus-transformed hamster glial cell line known as HJC-15. Two of them (PAb 416 and PAb 108) detected a nuclear antigen in both SV40-transformed CCL 75.1 cells and in HJC-15 cells, but not in control cells lacking T-antigen. These same antibodies also labeled a nuclear antigen in hamster tumor tissue derived from HJC-15 cells. In addition, the monoclonal antibody PAb 416 detected a nuclear antigen in progressive multifocal leukoencephalopathy (PML) tissue infected with JC virus, but not in normal brain tissue or tissue from other neurological diseases. Staining by PAb 416 was reduced by prior incubation with hamster anti-JCV tumor serum, suggesting that the polyclonal antiserum to JCV T-antigen may compete for an epitope at or near the PAb 416 binding site.

A double-label method detects both early (T-antigen) and late (capsid) proteins of JC virus in progressive multifocal leukoencephalopathy brain tissue from AIDS and non-AIDS patients.

A new double-label immunocytochemical method detects JC virus (JCV) early (T-antigen) and late (capsid) proteins simultaneously in cryostat sections of progressive multifocal leukoencephalopathy (PML) brain tissue from both acquired immunodeficiency syndrome (AIDS) and non-AIDS patients. T-antigen is detected with a monoclonal antibody (PAb 416) followed by goat anti-mouse IgG and mouse Clono-PAP, while capsid proteins are detected by a rabbit polyclonal antiserum to capsid proteins followed by biotinylated goat anti-rabbit IgG and streptavidin-alkaline phosphatase conjugate. The substrates are 3,3'-diaminobenzidine and Vector Red I, respectively. With this method some infected glial cells stain for late (capsid) antigens in the nucleus, while others show early protein (large T-antigen) immunoreactivity. The latter are likely to be astrocytes infected abortively or oligodendrocytes in the early stages of a productive JCV infection.

Myelin-associated glycoprotein (MAG) distribution in human central nervous tissue studied immunocytochemically with monoclonal antibody.

Recent biochemical data show that myelin-associated glycoprotein (MAG) is the antigen for a monoclonal antibody found in sera of patients with IgM paraproteinemia and neuropathy (Braun et al. 1982). Immunoreactivity of this antibody with CNS has not been described. To study this, monoclonal anti-MAG was used in the avidin-biotin-peroxidase complex method (Hsu et al. 1981) to immunostain paraffin and epon sections of human CNS. Well characterized polyclonal MAG antiserum (Quarles et al. 1981) was employed in comparison tests. In paraffin sections of developing CNS, both monoclonal and polyclonal MAG antisera stained oligodendroglia and myelin. In adult CNS, periaxonal regions of myelin sheaths were immunostained in paraffin sections and semithin epon sections treated with monoclonal and polyclonal anti-MAG. In electron-microscopic experiments that included milder pretreatment of epon thin sections and more precise reaction product localization, entire thickness of myelin sheaths were immunostained. Thus, in electron micrographs, monoclonal and polyclonal anti-MAG immunoreactivity also have the same localization. In other electron-microscopic experiments, the same reaction product localization was observed with antiserum to myelin basic protein (MBP), a known constituent of compact myelin. Thus, results with this monoclonal anti-MAG provide important new evidence to support the localization of MAG in compact CNS myelin. Our data also suggest that monoclonal antibodies against MAG will be useful in studies of the pathogenesis of multiple sclerosis and other demyelinating diseases.

Effects of platelet glycoprotein IIb/IIIa receptor blockade by a chimeric monoclonal antibody (abciximab) on acute and six-month outcomes after percutaneous transluminal coronary angioplasty for acute myocardial infarction. EPIC investigators.

Percutaneous transluminal coronary angioplasty (PTCA) for acute myocardial infarction is an attractive alternative to thrombolysis, but is still limited by recurrent ischemia and restenosis. We determined whether adjunctive platelet glycoprotein IIb/IIIa receptor blockade improved outcomes in patients undergoing direct and rescue PTCA in the Evaluation of c7E3 for Prevention of Ischemic Complications (EPIC) trial. Of the 2,099 patients undergoing percutaneous intervention who randomly received chimeric 7E3 Fab (c7E3) as a bolus, a bolus and 12-hour infusion, or placebo, 42 underwent direct PTCA for acute myocardial infarction and 22 patients had rescue PTCA after failed thrombolysis. The primary composite end point comprised death, reinfarction, repeat intervention, or bypass surgery. Outcomes were assessed at 30 days and 6 months. Baseline characteristics were similar in direct and rescue PTCA patients. Pooling the 2 groups, c7E3 bolus and infusion reduced the primary composite end point by 83% (26.1% placebo vs 4.5% c7E3 bolus and infusion, p = 0.06). No reinfarctions or repeat urgent interventions occurred in c7E3 bolus and infusion patients at 30 days, although there was a trend toward more deaths in c7E3-treated patients. Major bleeding was increased with c7E3 (24% vs 13%, p = 0.28). At 6 months, ischemic events were reduced from 47.8% with placebo to 4.5% with c7E3 bolus and infusion (p = 0.002), particularly reinfarction (p = 0.05) and repeat revascularization (p = 0.002). We conclude that adjunctive c7E3 therapy during direct and rescue PTCA decreased acute ischemic events and clinical restenosis in the EPIC trial. These data provide initial evidence of benefit for glycoprotein IIb/IIIa receptor blockade during PTCA for acute myocardial infarction.

Molecular genotyping of BK and JC viruses in human polyomavirus-associated interstitial nephritis after renal transplantation.

The human polyomaviruses BK virus (BKV) and JC virus (JCV) have been linked to ureteric stenosis and allograft interstitial nephritis, but molecular characterization of the species involved has not been performed. We studied paraffin-embedded renal tissue from 19 cases of allograft viral interstitial nephritis. Histological sections were subjected to polymerase chain reaction amplification using consensus, BKV-, and JCV-specific primers, with subsequent DNA sequencing for strain determination. BKV was present in all (100%) interstitial nephritis kidneys and placed in genotypes corresponding to serological groups I (n = 11), II (n = 1), and IV (n = 5). Fourteen of 17 isolates (82%) showed sequence variations in the viral capsid protein-1 (VP1) capsid region, with predicted changes in the encoded amino acids and sometimes with potential implications for the secondary and tertiary structure of the corresponding protein molecules. An additional case showed a previously reported glutamine-->leucine T-antigen region mutation. JCV was seen in seven interstitial nephritis kidneys (37%), with types 4 (n = 3), 3A (n = 2), and 2A (n = 1) identified. Most white individuals with asymptomatic infection are reported to shed type 1 JCV in the urine. Simian 40 polyomavirus was not identified in any case. These observations may have pathogenic relevance to the development of an extremely refractory form of polyomavirus interstitial nephritis seen after kidney transplantation.

The importance of urinary magnesium values in patients with gut failure.

OBJECTIVE: To determine whether urinary magnesium (Mg) values in patients with gut failure would be more helpful than serum Mg measurements in assessment of Mg deficiency. DESIGN: We compared serum and urinary Mg values in 16 patients with gut failure and 16 age- and sex-matched control subjects. MATERIAL AND METHODS: Sixteen patients with gut failure (nine women and seven men; mean age, 59 years) had serum and 24-hour urinary mg measured before Mg replacement therapy. Short bowel syndrome was present in 75%, and diffuse small bowel disease was present in 25%. RESULTS: The median value for serum Mg was 1.7 mg/dL for patients and 2.0 mg/dL for healthy control subjects (P < 0.001). The median values for urinary Mg were 19 mg and 127 mg per 24-hour specimen in patient and control groups, respectively (P < 0.001). A strong correlation was noted between serum Mg and urinary Mg levels. All patients had low urinary Mg values even though 9 of 16 (56%) had normal serum Mg values. Two patients with normal serum Mg concentrations had urinary Mg values of 20 mg/24 h (25% of normal). Serum, but not urinary, Mg correlated significantly with the length of remaining small bowel (P = 0.03). CONCLUSIONS: Urinary Mg declines before serum Mg and is an earlier and more reliable indicator of evolving Mg deficiency. On the basis of these observations and those showing beneficial effects of parenterally administered Mg supplements on urinary citrate excretion (and, presumably, formation of calcium oxalate stones), replacement of Mg in patients with gut failure should be targeted at normalizing urinary Mg.

Detection of JC virus in two African cases of progressive multifocal leukoencephalopathy including identification of JCV type 3 in a Gambian AIDS patient.

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating central nervous system (CNS) infection, affecting mainly oligodendrocytes, but also occasional astrocytes. In the USA, Europe and Asia, PML is caused by the human polyomavirus JC virus (JCV) and in autopsy series occurs in about 4-7% of AIDS patients. In Africa, the prevalence of PML in AIDS patients is uncertain and the causative agent is unknown. This study reports immunocytochemical and PCR confirmation of PML in the CNS of an AIDS patient dying in Uganda, East Africa (case 1). In a Gambian patient infected with HIV-2 who died 3 months after onset of AIDS/PML in Germany (case 2), it was possible to confirm the identity of the virus by DNA sequencing of the PCR amplified JCV product. This African genotype of the virus (type 3) showed an unusual re-arrangement of the regulatory region, and could be distinguished at several sites from East African and African-American JCV strains described previously. This study has confirmed that PML is a complication of African AIDS as it is in Europe and the USA, and that JCV type 3 is pathogenic in African AIDS patients. Furthermore, the finding of an African genotype of JCV in a patient dying in Germany suggests that in this individual JCV represented a latent infection acquired in Africa.

Tri-primer PCR can distinguish free and cloned viral DNA in a single reaction.

A polymerase chain reaction based method is described which distinguishes free and cloned JC virus (JCV) DNA in a single reaction. The method utilizes three primers and amplifies DNA fragments of two different sizes, one specific for free and another specific for pBR322 cloned viral DNA. The procedure was used to simultaneously detect the presence of free and contaminating cloned JCV DNA in brain tissues.

Type-specific amplification of viral DNA using touchdown and hot start PCR.

Two types of JC virus (JCV) are found in infected brain and kidney tissues. A highly reliable PCR assay to determine viral type in tissue is presented. This type-specific system is analogous to allele-specific PCR used to detect point mutations in cellular genes. Specific amplification of two fragments, using four pairs of type-specific primers, is based on a single nucleotide difference at the 3'-ends of the primers. A combination of three conditions in the PCR reaction was required for specificity: 'hot start', a ramped ('touchdown') cycle profile, and a slightly lowered molar concentration of the specific primers and dNTPs. Efficient yield of PCR product is not lost under these conditions, and even the least selective mismatches (C:A and T:G) provided specific amplification. Type-specific restriction enzyme sites within the amplified fragments confirm type designation.

Progressive multifocal leukoencephalopathy and JC virus genotypes in West African patients with acquired immunodeficiency syndrome: a pathologic and DNA sequence analysis of 4 cases.

OBJECTIVE: Progressive multifocal leukoencephalopathy is caused by polyomavirus JC in immunosuppressed patients. JC virus genotypes are identified by sequence analysis of the viral genome. Despite the prevalence of acquired immunodeficiency syndrome in sub-Saharan Africa, few cases of progressive multifocal leukoencephalopathy have been reported from this region. Here we describe 4 African cases and provide an analysis of viral genotypes. METHODS: Immunohistochemical staining by labeled streptavidin-biotin for capsid protein antigen was performed on all cases. Polymerase chain reaction amplification of viral genomic DNA was followed by direct cycle sequencing. RESULTS: JC virus type 3 was identified in 2 cases, and type 6 was isolated in 1 case. The viral regulatory region from 1 case showed an uncommon rearrangement pattern. CONCLUSIONS: Progressive multifocal leukoencephalopathy in West African patients with acquired immunodeficiency syndrome is caused by African genotypes of JC virus (types 3 and 6). The prevalence of disease in this autopsy series from sub-Saharan Africa (1.5%) was less than has been reported from Europe and the United States (4% to 10%) and may be partly due to biological differences in JC virus genotypes. Further studies will be needed to confirm this observation.

Conservation throughout vertebrate evolution of the predicted beta-strands in myelin basic protein.

To identify functionally important parts of the 18.5-kDa myelin basic protein (MBP), the amino acid sequences from 10 species ranging from shark to human were aligned using the SEQHP computer program. The residues that are invariant or very conservatively substituted (Arg/Lys, Ser/Thr, Ile/Leu, Asp/Glu) among all 10 proteins were scored. Of the 72 conserved residues in the 170-residue human protein (42% conserved), 32 are found within the five beta-strands previously predicted (45 residues, 71% conserved), 23 within the small-loops region (42 residues, 55% conserved), but only 17 within the large-loops region (83 residues, 20% conserved). Of the 22 hydrophobic residues within the predicted beta-sheet of human MBP, 20 hydrophobic residues remain in the shark protein, 19 of them in the same positions. In contrast, there are 10 hydrophobic residues elsewhere in the human protein, but only 7 remain in the shark protein and only 1 of them is in the same position. The triprolyl sequence found in all mammalian MBPs and in the chicken MBP is not conserved in the shark protein. The four alternately spliced forms of mouse MBP can be accommodated by the beta-structural model, but not the 17-kDa human MBP, which lacks exon 5. These findings confirm the crucial role of the hydrophobic residues in the predicted beta-sheet for the structure and function of the protein. It seems likely that the conserved portions of the protein make an important contribution to the highly ordered lamellar structure of myelin.