Molecular evaluation of BK polyomavirus nephropathy.

Understanding at a molecular level, the immunologic response of polyomavirus nephropathy (PVN), a critical cause of kidney graft loss, could lead to new targets for treatment and diagnosis. We undertook a transcriptional evaluation of kidney allograft biopsies from recipients with PVN or acute rejection (AR), as well as from recipients with stable allograft function (SF). In both the PVN and AR groups, Banff histologic scores and immunohistochemical analysis of inflammatory infiltrates were similar. Despite their different etiologies, the transcriptional profiles of PVN and AR were remarkably similar. However, transcription of genes previously linked to AR including CD8 (65.9 +/- 18.8) and related molecules IFN-gamma(55.1 +/- 17.0), CXCR3 (49.9 +/- 12.8) and perforin (153.8 +/- 50.4) were significantly higher in PVN compared to AR (30.9 +/- 2.0, 14.0 +/- 7.3, 12.1 +/- 7.3 and 15.6 +/- 3.8-fold, respectively; p < 0.01). Importantly, transcription of molecules associated with graft fibrosis including matrix collagens, TGFbeta, MMP2 and 9, as well as markers of epithelial-mesenchymal transformation (EMT) were significantly higher in PVN than AR. Thus, renal allografts with PVN transcribe proinflammatory genes equal in character and larger in magnitude to that seen during acute cellular rejection. BK infection creates a transcriptional microenvironment that promotes graft fibrosis. These findings provide new insights into the intrarenal inflammation of BK infection that promotes graft loss.

Relation of JC virus DNA in the cerebrospinal fluid to survival in acquired immunodeficiency syndrome patients with biopsy-proven progressive multifocal leukoencephalopathy.

The detection and semiquantitation of JC virus (JCV) DNA in cerebrospinal fluid (CSF) is prognostic of survival and is a marker of the course of progressive multifocal leukoencephalopathy (PML). CSF samples from 15 acquired immunodeficiency syndrome (AIDS) patients with biopsy-proven PML were analyzed by semiquantitative polymerase chain reaction (PCR). A low JCV burden was predictive of longer survival compared with a high JCV burden (median survival from entry, 24 [2-63] vs 7.6 [4-17] weeks). Further analyses indicated a possible threshold of 50 to 100 copies/microl separating high- and moderate-risk cases. Patients with a JCV load below this level survived longer than those with a JCV load above it.

Hypothesis: "Rogue cell"-type chromosomal damage in lymphocytes is associated with infection with the JC human polyoma virus and has implications for oncopenesis.

The hemagglutination inhibition antibody titers against the JC and BK polyoma viruses (JCV and BKV, respectively) are significantly elevated in individuals exhibiting "rogue" cells among their cultured lymphocytes. However, the elevation is so much greater with respect to JCV that the BKV elevation could readily be explained by cross reactivity to the capsid protein of these two closely related viruses. The JCV exhibits high sequence homology with the simian papovavirus, simian virus 40 (SV40), and inoculation of human fetal brain cells with JCV produces polyploidy and chromosomal damage very similar to that produced by SV40. We suggest, by analogy with the effects of SV40, that these changes are due to the action of the viral large tumor antigen, a pluripotent DNA binding protein that acts in both transcription and replication. The implications of these findings for oncogenesis are briefly discussed.

Improved electrochemiluminescent label for DNA probe assays: rapid quantitative assays of HIV-1 polymerase chain reaction products.

We describe the characterization and utility of a new electrochemiluminescent (ECL) label for oligonucleotides, utilizing phosphoramidite chemistry. This phosphoramidite of the tris(2,2-bipyridine)ruthenium(II) complex, bis(2,2-bipyridine)(4-[4-(2-cyanoethoxy-N,N-diisopropyl-amino) phosphinoxybutyl]4'-methyl)2,2-bipyridine ruthenium(II) dihexafluorophosphate or Origen phosphoramidite, enables the direct incorporation of the label during automated DNA synthesis. Efficiency of this automated synthesis allows the direct utilization of probes without further purification. Introduction of this labeling group is reproducible, and the ECL signal recovered is not influenced by hybridization. Furthermore, neither hybridization kinetics nor hybrid stability was affected by our label. We also demonstrate the utility of these labels for the development of rapid assays with oligonucleotides direct from automated synthesis. The clinical utility of these labeled oligonucleotides is shown with assays of total nucleic acid, extracted from peripheral blood lymphocytes of patients with acquired immunodeficiency syndrome (AIDS), to detect the human immunodeficiency virus (HIV-1). The results demonstrate the ability of the assay to quantify 30-2000 copies of HIV1 gag genes and to rapidly detect (less than 45 min) HIV-1 gag genes in a nonseparation assay. The application of this assay to clinical samples demonstrates the utility of these assays for rapid and quantitative analysis.

Detection of JC virus DNA in peripheral lymphocytes from patients with and without progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) results from lytic infection of oligodendrocytes by JC virus (JCV). Although JCV has been identified in mononuclear cells in bone marrow and hematogenous dissemination of the virus to the central nervous system has been suspected, JCV has never been clearly demonstrated in the peripheral circulation. Using polymerase chain reaction technology, we examined peripheral lymphocytes of 19 patients with brain biopsy-proven PML for the JCV genome. Two non-PML control groups, consisting of 26 patients seopositive for human immunodeficiency virus type 1 (HIV-1) and 30 immunocompetent patients with Parkinson's disease, were also examined for the presence of the JCV genome in lymphocytes. Cerebrospinal fluid from 10 patients with PML was examined for the presence of the JCV genome as well. The JCV genome was detected in the lymphocytes of 89% (17) of the patients with PML, 38% (10) of the HIV-1-seropositive patients without PML, and none of the patients with Parkinson's disease. Sequencing of the JCV regulatory region from the lymphocytes of three patients revealed the prototype MAD-1 strain of JCV in one patient with PML, a MAD-4 strain in a second patient with PML, and a slightly modified MAD-4 strain in an HIV-1-positive patient without PML. Only 3 of 10 patients with PML who had JCV detected in lymphocytes had the JCV genome in their cerebrospinal fluid. These results demonstrate that the JCV genome can be found in circulating lymphocytes from patients with PML and suggest that lymphocytes are an important vector for hematogenous dissemination of JCV to the central nervous system.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental congenital disease with simian cytomegalovirus in rhesus monkeys.

Cytomegalovirus (CMV) infections occur worldwide and are responsible for severe damage to the child in from one to five newborns per 20,000 births. Animal models of congenital CMV infection resulting in disease have been developed in mice and guinea pigs. We report here the development of ventricular dilatation and leptomeningitis in rhesus monkeys, Macaca mulatta, following intrauterine infection with rhesus cytomegalovirus (RCMV). Central nervous system (CNS) lesions were associated with low cytomegalovirus fluorescent antibody titers in affected fetuses. In several infected animals, RCMV was isolated at necropsy from neural and nonneural tissues taken shortly after birth. This model allows investigators to study the pathogenesis and prevention of CNS changes following RCMV infection.

Coxsackie virus B4 produces transient diabetes in nonhuman primates.

Cynomolgus, rhesus, and Cebus monkeys failed to show glucose tolerance or insulin secretion abnormalities after infection with encephalomyocarditis virus or Coxsackie virus B4. Patas monkeys also showed no abnormalities after infection with encephalomyocarditis virus. However, patas monkeys infected with Coxsackie virus B4 or treated first with a subdiabetogenic dose of streptozocin and then infected sequentially with Coxsackie viruses B4 and B3 showed transient elevation of glucose tolerance tests, depressed insulin secretion, and glucose in the urine. Our experiments in nonhuman primates support earlier studies in mice and humans that under certain circumstances, Coxsackie viruses can cause abnormalities in glucose homeostasis.

Serum antibody responses of juvenile and infant rhesus monkeys injected with Haemophilus influenzae type b and pneumococcus type 6A capsular polysaccharide-protein conjugates.

Juvenile and infant rhesus monkeys were injected subcutaneously with saline solutions of Haemophilus influenzae type b (Hib) and pneumococcus type 6A (Pn6A) capsular polysaccharides conjugated to either tetanus toxoid (TT), horseshoe crab hemocyanin, or cholera toxin (CT), and the antibody responses of the monkeys to both bacterial components were measured. All three Hib conjugates were immunogenic and elicited booster responses; their comparative immunogenicity was Hib-CT greater than Hib-TT greater than Hib-horseshoe crab hemocyanin. Hib alone did not elicit antibodies in the juveniles. Juveniles responded earlier and with higher levels of antibodies than did infants. TT, as well as diphtheria-tetanus toxoids-pertussis vaccine adsorbed injected concurrently at a separate site, increased both Hib and TT antibody responses in juveniles (P less than 0.05). Concurrent injection of 5 Lf of fluid TT with a nonimmunogenic 5-micrograms dose in infants elicited levels of Hib antibodies comparable to those elicited by 50 micrograms of Hib-TT. Hib antibodies elicited by the conjugates remained at protective levels in both juveniles and infants 2 months after the last injection, were bactericidal, and conferred passive immunity against bacteremia in infant rats. Passive immunization of juveniles with tetanus immune globulin before each injection of Hib-TT did not suppress Hib antibodies. Hib-TT and Hib-CT elicited increases of Hib antibodies of the immunoglobulin M and G isotypes in the infants. The Pn6A-TT conjugate was considerably less immunogenic than the Hib-TT conjugate; only a few of the juveniles or infants responded with protective levels of Pn6A antibodies. Pn6A antibodies from responders conferred protection in mice against intraperitoneal challenge with Pn6A organisms. TT antibodies were elicited in both juvenile and infant animals after one injection of 50 micrograms of Hib-TT and in the infants injected with 5 micrograms of Hib-TT plus 5 Lf of TT; 5 micrograms of Hib-TT and Pn6A-TT in combination alone did not elicit TT antibodies. Hib-CT elicited CT antibodies in both juveniles and infants.

Intravenous immunoglobulin in neonatal group B streptococcal disease. Pharmacokinetic and safety studies in monkeys and humans.

Numerous studies have suggested that opsonic antibody is important in neonatal immunity to group B streptococci. Immunoglobulin G is primarily transferred from the mother to the fetus across the placenta in the last few weeks of pregnancy. Premature babies may, therefore, not acquire sufficient opsonic antibody to protect them from infection with group B streptococci. Although maternal immunization may provide adequate maternal opsonic antibody, premature infants with antibody deficiency may remain susceptible to infection. Intravenous immunoglobulin administered to term pregnant rhesus monkeys did not provide reliable levels of serum opsonic activity to group B streptococci in their offspring. Pharmacokinetic and safety studies were also performed in human neonates. Significant elevations in group B streptococcal-specific IgG did occur in human neonates given 500 mg/kg intravenous immunoglobulin and the infusions appeared safe and well tolerated. The availability of intravenous immunoglobulin with functional activity against group B streptococci may provide a rapid and effective method of delivering opsonic antibody to neonates.

Antibody to type III group B Streptococcus in the rhesus monkey.

Susceptibility to infection due to intra-amniotic type III group B streptococcal infection was studied in 27 rhesus monkeys. Sera from mothers and their offspring were tested to determine the concentration of antibody to the native type III group B Streptococcus antigen. Among 17 controls there was a statistically significant association between the concentration of maternal antibody prior to infection and both the neonatal survival rate and survival time (P less than 0.05). Neonatal survival was decreased to less than or equal to 6 hours (P = 0.005) if the maternal antibody concentration was less than 0.5 micrograms/ml. Modified immune serum globulin was given intravenously to the mothers prior to intra-amniotic infection with (five animals) or without (five animals) neonatal modified immune serum globulin. Neither of the modified immune serum globulin groups demonstrated a significant reduction in the neonatal mortality rate; however, the addition of the modified immune serum globulin provided protection against rapid neonatal death among those animals born to mothers which had low or no detectable antibody. All maternal groups developed a significant increase in the concentration of antibody in postpartum sera. These results indicate that both naturally acquired and passive (modified immune serum globulin) antibodies to type III group B Streptococcus antigen are partially protective against intra-amniotic infection.

Detection of group B streptococcal antigens in amniotic fluid of rhesus monkeys.

To simulate group B streptococci (GBS) amniotic fluid infections common in humans and to examine bacterial growth and the appearance of GBS antigens in vivo, GBS were injected into the amniotic cavity of 19 near-term rhesus monkeys. Transabdominal aspirates of amniotic fluid were obtained before bacterial challenge, after 2 and 6 h, and during cesarean section delivery (24 h). Each fluid was quantitatively cultured for GBS. Specimens of amniotic fluid and gastric aspirate from each infant were tested for the presence of GBS antigens with a commercial latex particle agglutination test (Wellcogen Strep B; Wellcome Diagnostics, Dartford, England). To eliminate nonspecific latex particle agglutination reactivity, presumably caused by proteins, a processing procedure was required. Despite active proliferation of bacteria, only 12% of the 2-h amniotic specimens were latex particle agglutination positive. In contrast, 94% of th3 6-h and 100% of the 24-h specimens had detectable antigens, as did 89% of the gastric fluid specimens aspirated from the 19 newborns. Latex particle agglutination tests, after proper processing, will readily detect GBS antigens in amniotic or gastric aspirate fluid from experimentally infected rhesus monkeys.

Teratological effects of western equine encephalitis virus on the fetal nervous system of Macaca mulatta.

Fetal rhesus monkeys were inoculated intracerebrally with an attenuated strain of western equine encephalitis virus. All animals developed microcephaly. Twelve of sixteen monkeys developed ex vacuo hydrocephalus. All virus inoculated fetuses developed WEE virus antibody. Virus could not be recovered at the time of delivery. Monkeys with the highest WEE antibody titers showed the greatest degree of hydrocephalus.

Intraamniotic infection due to group G streptococcus: treatment and antibody response.

Penicillin treatment and antibody response were studied using a rhesus monkey model for intraamniotic infection with type III group B streptococci (T3GBS). Acute and convalescent phase sera from mothers and their offspring were tested with a radioactive antigen-binding assay to determine the concentration of antibody to the capsular T3GBS antigen. The frequency of placentitis was significantly lower in penicillin-treated animals (3 of 8) than in controls (10 of 10; P less than .01). The penicillin group also had a significantly lower neonatal mortality (1 of 9) than controls (6 of 10; P less than .05). Both groups of rhesus mothers developed a significant increase in concentration of antibody to T3GBS, but the antibody response was of lesser magnitude in the penicillin-treated group. This experimental model appears to be useful for studying both therapy for intraamniotic infection and the humoral immune response to infection with T3GBS.

Penicillin treatment for group B streptococcal meningitis in the rhesus monkey.

Penicillin therapy for experimentally produced neonatal meningitis due to intracerebral inoculation of group B streptococci (GBS) was studied in 25 rhesus monkeys. Penicillin was administered either therapeutically to the newborns 3 hours after GBS inoculation or prophylactically as a bolus to the pregnant females 2 hours before delivery. The neonatal mortality in the newborn treatment groups was 40% (6 of 15) compared to 100% (5 of 5) in the maternal prophylaxis group, and 0% (0 of 5) among uninfected and untreated controls. It was concluded that although penicillin can be used successfully to treat neonates with meningitis after intracerebral inoculation of GBS, penicillin given antepartum as bolus prophylaxis to the mother monkey was ineffective.

Experimental group B streptococcal infection in the rhesus monkey. I. Disease production in the neonate.

Group B streptococci (GBS) are responsible for serious infections of newborn infants. An experimental model for GBS infection was developed in the newborn rhesus monkey in order to obtain more information concerning the pathogenesis of such infections. A series of 29 newborn monkeys were inoculated with either type Ic or type III GBS or sterile broth. Fatal neonatal meningitis without associated pneumonia was produced consistently following intracerebral inoculation with either type Ic or type III; intracerebral inoculation with sterile broth produced no apparent disease. Variable disease production followed intravenous or intra-amniotic GBS inoculation, and clinical manifestations ranged from no apparent disease to fatal meningitis and pneumonia. This monkey model may be useful for further investigation of treatment and prevention of neonatal GBS infection.