Vertical Distribution of Rotylenchulus reniformis in Cotton Fields.

The possible impact of Rotylenchulus reniformis below plow depth was evaluated by measuring the vertical distribution of R. reniformis and soil texture in 20 symptomatic fields on 17 farms across six states. The mean nematode population density per field, 0 to 122 cm deep, ranged from 0.4 to 63 nematodes/g soil, and in 15 fields more than half of the R. reniformis present were below 30.5 cm, which is the greatest depth usually plowed by farmers or sampled by consultants. In 11 fields measured, root density was greatest in the top 15 cm of soil; however, roots consistently penetrated 92 to 122 cm deep by midseason, and in five fields in Texas and Louisiana the ratio of nematodes to root-length density within soil increased with depth. Repeated sampling during the year in Texas indicated that up to 20% of the nematodes in soil below 60 cm in the fall survived the winter. Differences between Baermann funnel and sugar flotation extraction methods were not important when compared with field-to-field differences in nematode populations and field-specific vertical distribution patterns. The results support the interpretation that R. reniformis below plow depth can significantly impact diagnosis and treatment of cotton fields infested with R. reniformis.

Ontogeny of androgen receptor immunoreactivity in lumbar motoneurons and in the sexually dimorphic levator ani muscle of male rats.

We documented the ontogeny of androgen receptor (AR) immunoreactivity for rat lumbar motoneurons of the sexually dimorphic motor pools, the spinal nucleus of the bulbocavernosus (SNB) and the dorsolateral nucleus (DLN), and for the sexually monomorphic retrodorsolateral nucleus (RDLN). We also assessed the ontogeny of AR immunoreactivity in the rat sexually dimorphic levator ani (LA), which is a target muscle for SNB motoneurons. Lumbar spinal cords and LA muscles from gonadally intact males at ages postnatal days (P)7, P10, and P14 and as adults were incubated with the rabbit antiserum PG-21. Half of the prepubertal males (P7-P14) received 200 micrograms of testosterone propionate (TP) 2 hours prior to death to enhance immunodetection of ARs. We found that SNB motoneurons developed AR immunoreactivity at first and achieved adult levels by P10. In contrast, the number of RDLN motoneurons with AR-immunopositive nuclei during development remained well below the adult number. Development of AR immunoreactivity in the DLN shared characteristics with both the SNB and the RDLN. AR immunoreactivity developed in some DLN motoneurons by P10, although the percentage of labelled motoneurons remained below that in adulthood. Acute TP treatment significantly increased the number of SNB motoneurons with AR-positive nuclei at P7. The LA showed a robust pattern of AR immunostaining from P7 to adulthood. Immunostaining was present only in nuclei and constituted only a subpopulation of the nuclei present in muscle. The present results confirm and extend previous results based on steroid autoradiography and steroid binding assays regarding regional and developmental differences in the expression of ARs.

Distribution of androgen receptor immunoreactivity in the spinal cord of wild-type, androgen-insensitive and gonadectomized male rats.

The polyclonal antiserum PG21 was used to detect androgen receptor (AR) protein in three motoneuronal pools of the male rat lumbar spinal cord. In gonadally intact, wild-type males, the spinal nucleus of the bulbocavernosus (SNB), dorsolateral nucleus (DLN), and retrodorsolateral nucleus (RDLN) all displayed immunolabeling of cell nuclei. The percentage of motoneurons displaying such labeling was highest in the SNB and lowest in the RDLN. This pattern of AR immunocytochemical labeling agrees well with previous steroid autoradiographic studies of androgen accumulation in the rat spinal cord. In contrast, virtually no motoneurons in any of the three pools displayed nuclear AR immunostaining in long-term gonadectomized males or in gonadally intact males carrying the Tfm mutation, which renders the AR incompetent. In gonadectomized males, labeling was restored in the SNB and DLN, but not the RDLN, 30 min after an injection of replacement testosterone. Eight hours of testosterone exposure restored immunolabeling in all three motor nuclei. Apparent cytoplasmic staining was seen in SNB motoneurons of untreated castrates and Tfm rats, but not intact rats, suggesting that AR residing in the cytoplasm translocates to the nucleus on binding to androgen in these motoneurons.

Brain tumors of owl monkeys inoculated with JC virus contain the JC virus genome.

The DNA from astrocytomas that developed in adult owl monkeys 16 to 36 months after intracranial inoculation with JC virus (JCV) was examined for the presence of the JCV genome by hybridization to cloned JCV DNA. The JCV genome was found to be integrated into the cellular DNA in all tumors examined. There was no JCV DNA in normal, uninvolved brain tissue from the same animals. Integration of the genome occurred at a limited number of sites in the cellular DNAs, indicating a clonal origin for the tumors, but none of the tumors had integration sites in common. In all but one of the tumors, there was tandem, head-to-tail integration of two or more copies of the JC genome. In a tumor which had only one integration site and could be analyzed more extensively, there appeared to be a complete copy of the JCV genome present, although deletions of small portions of the genome would not have been detected.

Characterization of tissue culture-induced heterogeneity in DNAs of independent isolates of JC virus.

After several serial passages at low multiplicities of infection in primary human foetal glial cells at 37 degrees C, the DNA of prototype (MAD-1) JC virus and that of MAD-2 and MAD-3 are typically heterogeneous in size, but DNAs of MAD-4 and MAD-6 are relatively homogeneous. A similar dichotomy was observed in the DNAs of six isolates propagated more recently in glial cultures at 39 degrees C under similar conditions of brief passage in vitro at low multiplicities of infection: the DNAs of two (MAD-9 and -10) were heterogeneous, but the DNAs of four others (MAD-8, -11, -12 and -14) were homogeneous. Therefore, the propensity of the viral genome to sustain deletions was an intrinsic property of each isolate. However, actual induction and maintenance of the presumably defective DNAs was influenced by the relative proportions of permissive spongioblasts and semi-permissive astrocytes in the glial cultures and by the multiplicity of infection. Deletions in MAD-1 DNA were confined to the presumptive early region and spanned the BamHI cleavage site (map position 0.505). The heterogeneity was more complex in the DNAs of MAD-2 and MAD-3, but again most of the deletions, which ranged up to 12% of full-length DNA, spanned the BamHI site. We propose that the differential susceptibility to deletion among isolates is a consequence of natural genetic variation in JC virus.

Tubulo-interstitial nephritis associated with polyomavirus (BK type) infection.

We studied viral injury to the kidney in a six-year-old boy with hyperimmunoglobulin M immunodeficiency who presented with irreversible acute renal failure and eventually died after five months of dialysis. Renal biopsy at the time of his presentation revealed a predominantly tubulo-interstitial process with numerous viral inclusions that were identified as polyomavirus. Urine cultures showed a massive viruria with BK-type, polyomavirus. The kidney disease was end stage, with persistence of BK virus identified by morphologic techniques and by culture. DNA hybridization analysis showed virus in low concentration in the lymph nodes, spleen, and lungs. The marked viruria, the high concentration of BK virus, and the extensive distribution of viral antigen throughout the kidney all suggest that infection with BK virus was the basis of the severe renal parenchymal injury.

Progressive multifocal leukoencephalopathy: a burnt-out case.

A patient with Hodgkin's disease developed progressive multifocal leukoencephalopathy (PML), documented by brain biopsy to be associated with JC virus infection. His disease progressed over several months, resulting in severe neurological deficit, but then stabilized with little or no further clinical progression during the remaining year of his life. Histopathological evaluation of the brain at autopsy supported the clinical impression that brain infection was arrested. Whereas the brain biopsy exhibited the histological features of active PML including giant bizarre astrocytes, at postmortem examination brain lesions appeared inactive, with regression of astrocytic changes and elimination of oligodendroglial inclusions. Similarly, JC virus antigen, present in the brain biopsy, was not detected in the autopsied brain. This case provides further evidence that PML is not invariably fatal and that clinical and cytological remission can occur.

Distribution of nonintegrated DNA from JC papovavirus in organs of patients with progressive multifocal leukoencephalopathy.

Tissues from 10 patients with progressive multifocal leukoencephalopathy (PML) and 14 patients without PML who were serologically positive for JC papovavirus were examined by molecular hybridization for human polyomavirus DNA sequences. Although viral proteins were not identified by fluorescent antibody methods, viral DNA was found in the kidneys of seven of nine patients with PML by hybridization, at 0.2-10 viral genome copies per cell genome equivalent, compared with 0.6-4 X 10(3) copies per cell in diseased areas of the brain. Examination of viral DNA from brains and kidneys of patients with PML by blot hybridization yielded no evidence of integration into the host cell genome. In three of the patients with PML, viral DNA was also found in liver, lung, lymph node, and spleen. Two of these patients, with widely disseminated JC virus, were children. In tissues from patients without PML, no evidence of JC virus infection was found, but BK papovavirus DNA was detected in two of 14 kidneys tested.

Virologic and serologic studies of progressive multifocal leukoencephalopathy.

The human polyomavirus JCV has been associated with diseased brain tissue from 54 cases of PML. The disease occurred in persons of all ages--the youngest being 5 years old--whose immune mechanisms were depressed for any of a variety of reasons. In six patients the disease ceased progressing and they stabilized or improved. The brains of two of these, who died months or years later, were found to be free of active PML at autopsy. Sera from 21 patients had HAI antibodies against JCV, but none had antibodies against T or the common internal antigens, and only one had IgM antibodies detectable by immunofluorescent staining. Nine CSF were devoid of JCV-related antibodies except for one which gave an HAI titer of 8. Virus has been isolated from 15 PML brains, and preliminary studies indicate that one isolate, while clearly of JCV type, is serologically distinguishable from prototype Mad 1 JCV.

New world primates as a model of viral-induced astrocytomas.

Owl and squirrel monkeys are susceptible to the oncogenic effects of JCV. These species of New World monkeys can be safely inoculated intracerebrally. Care must be taken with owl monkeys since they have an inherited clotting abnormality. Incubation times for the development of tumors range from 14 to 30 months. Anorexia was the first clinical sign of tumor development. The clinical course is rapid with death within two to three days. This model provides a means for studying diagnostic, virological, immunological and therapeutic techniques which are applicable to human patients with astrocytomas.

Viral-induced astrocytomas in squirrel monkeys.

Saimiri sciureus, the squirrel monkey, is susceptible to the oncogenic effects of JCV following intracerebral inoculation. As in owl monkeys, tumor development follows an incubation time of 14 to 30 months. Four of six virus-inoculated monkeys developed cerebral tumors, three of which were astrocytomas grade 4 and the remaining tumor was a poorly differentiated astrocytoma. All tumors showed high cellularity, mitotic figures, and cellular pleomorphism. In the astrocytoma grade 4, neovascularization was a prominent feature. The blood vessels in the poorly differentiated astrocytoma appeared normal. Multinucleated giant cells were present in all four astrocytomas. Antemortem hemorrhage was seen in one astrocytoma grade 4. Other tumor types were not seen. The occurrence of astrocytomas in a second species of New World monkeys confirms the oncogenicity of JCV for nonhuman primates.

Glial and divergent cells in primate central nervous system tumors induced by JC virus isolated from human progressive multifocal leukoencephalopathy (PML).

Immunofluorescent stains for fibronectin (FN) and glial fibrillary acidic protein (GFAP) were used in conjunction with routine histologic stains to study tumors induced in squirrel and owl monkeys by JC virus from progressive multifocal leukoencephalopathy (PML). Three varieties of glioma were identified. The first and most common variety was a neoplasm similar to grade 4 astrocytoma in humans. The second had thin, normal-appearing FN-positive vessel walls and a vastly expanded neuroectodermal parenchyma which could not be characterized by routine histologic stains. Anti-GFAP revealed the glial nature of the parenchyma. Isolating glial parenchymal cells from divergent FN-positive cells has become important to neurooncology. This type of tumor may be of particular interest for such isolations due to its high ratio of glial cells to divergent cells. The third variety was not a homogeneous neoplasm. It occurred as focal regions within tumors of the first type, and consisted of giant cells with huge nuclei. These cells resemble the cells of a human giant cell glioblastoma and bear a slight similarity to the bizarre glial cells seen in PML. The rare human giant cell glioblastoma might have an association with JC virus or with PML.

Neuroradiological studies of JCV-induced astrocytomas in nonhuman primates.

Astrocytomas in nonhuman primates following JC virus inoculation provides a model which can be used to evaluate diagnostic and therapeutic techniques used in humans. The CT scan appearance of astrocytomas in nonhuman primates closely resembles that seen in humans. Our studies have shown that tumors may be detected in asymptomatic monkeys. Serial scans have shown astrocytomas to grow rapidly with breakdown of the blood-brain barrier. CT scanning has demonstrated the presence of tumor which was undetectable by gross examination at necropsy but confirmed by light microscopy. Studies are in progress to further define the radiological appearance of gliomas, to evaluate contrast-tagged anti-tumor antibodies as a diagnostic tool in evaluating gliomas by computerized tomography, and to evaluate metabolic parameters of actrocytomas by positron emission tomography.

The detection of JC viral genome in owl monkey tumors.

The JC viral genome is integrated into the cellular DNA of brain tumors from owl monkeys inoculated intracranially with JC virus 16-36 months prior to the development of the tumors. Normal brain tissue of the animals did not contain the viral genome. For at least two tumors, restriction endonuclease analysis of the tumor DNA indicates that there is a complete copy of the viral genome present. Integration of the genome in all but one of the tumors was at only one or only a limited number of sites, indicating a clonal origin for the tumors. In all but one of the tumors, there was tandem, head-to-tail integration of two or more copies of the JC genomes.

Survey of human polyomavirus (JCV, BKV) infections in 139 patients with lung cancer, breast cancer, melanoma, or lymphoma.

In 139 patients with breast or lung carcinoma, malignant melanoma, and Hodgkin's or non-Hodgkin's lymphoma, JC and BK virus serology (hemagglutination inhibition) and urinary polyomavirus excretion (cytology and immunofluorescence microscopy) were studied. Overall, 18 of 70 patients with paired sera (26%) had titer increases against JC or BK virus, and 11 of 114 patients (10%) had evidence for urinary excretion. The infection rate was highest in patients with Hodgkin's or non-Hodgkin's lymphoma (approximately 40%). Serum HAI antibody titers against JC and BK viruses appeared similar to age-matched controls in each patient group--with the exception of decreased BK titers at diagnosis in patients with resected malignant melanoma and increased JC virus antibody titers at diagnosis in patients with poor-prognosis non-Hodgkin's lymphoma (IC-2). The biologic significance of these observations remains to be determined. Initial antibody titers against JC or BK virus were not of prognostic value for subsequent survival in any of the tested patient groups. Both nonspecific immunotherapy and aggressive, multidrug chemotherapy had surprisingly little effect on serum HAI titers to JC or BK virus. Patients with poor-prognosis non-Hodgkin's lymphoma appear especially suitable for further investigation of JC and BK virus infections. Study of nonbrain, nonurinary-tract tissues may disclose other parenchymal sites of polyomavirus replication in these patients.

Naturally occurring and passage-induced variation in the genome of JC virus.

We have examined both the naturally occurring and passage-induced variation in the genome of the human polyomavirus, JCV. JCV DNA was extracted directly from diseased brain tissue of ten cases of progressive multifocal leukoencephalopathy (PML) and the DNA population from any one case was homogeneous with respect to length and restriction endonuclease cleavage patterns. However, a comparison of cloned JCV DNAs revealed that the viral DNAs derived from different cases of PML were not identical. We identified a hypervariable region near the origin of DNA replication, and we demonstrated that there are two genetically distinguishable subtypes of the virus. After growth in vitro, the DNA from certain isolates of JCV became heterogeneous in size. Deletions of up to 12% of the genome occurred after as few a three low multiplicity passages in human glial cells. Most of the deletions mapped within the region presumed to code for T antigen. In addition, we examined the physical properties of JCV from brain and, as expected, they were typical of the polyomavirus genus.