Low-level monocyte chemoattractant protein-1 stimulation of monocytes leads to tumor formation in nontumorigenic melanoma cells.

Tumors commonly produce chemokines for recruitment of host cells, but the biological significance of tumor-infiltrating inflammatory cells, such as monocytes/macrophages, for disease outcome is not clear. Here, we show that all of 30 melanoma cell lines secreted monocyte chemoattractant protein-1 (MCP-1), whereas normal melanocytes did not. When low MCP-1-producing melanoma cells from a biologically early, nontumorigenic stage were transduced to overexpress the MCP-1 gene, tumor formation depended on the level of chemokine secretion and monocyte infiltration; low-level MCP-1 secretion with modest monocyte infiltration resulted in tumor formation, whereas high secretion was associated with massive monocyte/macrophage infiltration into the tumor mass, leading to its destruction within a few days after injection into mice. Tumor growth stimulated by monocytes/macrophages was due to increased angiogenesis. Vessel formation in vitro was inhibited with mAbs against TNF-alpha, which, when secreted by cocultures of melanoma cells with human monocytes, induced endothelial cells under collagen gels to form branching, tubular structures. These studies demonstrate that the biological effects of tumor-derived MCP-1 are biphasic, depending on the level of secretion. This correlates with the degree of monocytic cell infiltration, which results in increased tumor vascularization and TNF-alpha production.

Astrocytes as targets for Venezuelan equine encephalitis virus infection.

Venezuelan equine encephalitis virus (VEE) produces an acute infection in humans and induces a well-characterized cytopathic effect in neurons of the central nervous system (CNS). However, little is known about the role of glial cells in response to VEE infection of the CNS. Our results demonstrate that VEE is capable of a productive infection in primary astrocyte cultures and that this infection is cytotoxic. Further, there were significant differences in the growth kinetics comparing virulent and attenuated strains of VEE. Additionally, VEE infection of astrocyte cultures induced gene expression of two neuro-immune modulators, tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS). Assays for TNF-alpha protein and nitric oxide (NO) demonstrated high levels of TNF-alpha protein and low levels of NO in response to VEE infection of astrocytes. These observations suggest an important role of astrocytes in this virus-induced encephalitis, and that interactions between astrocytes, other glial cells, and neurons may be important in VEE pathogenesis. Such interactions, which could impact neuronal survival, may include loss of functional changes in astrocytes or, alternatively, their production of neurotoxic molecules.

How to establish a perinatal home care program.

Current clinical data indicates that despite advances in the obstetric and neonatal arenas, preterm birth and subsequent low birth weight infants continue to be born. Technology-based perinatal home care programs are the fastest growing segment of services available to high-risk pregnant women, recuperating post-partum women, and newborns. It is anticipated that these services will alter forever the traditional measures of care for high-risk pregnant women and newborns.

Host range determinant in the late region of SV40 and RF virus affecting growth in human cells.

WtSV40 and its variant EL-SV40 (contains two complementing defective genomes) fail to productively infect human embryonic kidney cells or human fibroblasts. However, early SV40 (E-SV40) genomes can propagate in human cells when complemented by a particular late RF virus (L-RFV) genome or the closely related wtBKV genome. The L-RFV genome (L-RFV clone H) contains a deleted early region, a complete set of BKV capsid genes, and a single SV40 regulatory region (acquired by recombination). In contrast, it was not possible to make the reciprocal genome cross in human cells; late SV40 genomes containing a deleted early region do not complement early RFV or early BKV DNAs. The L-RFV clone H genome was also shown to complement wtSV40 in human cells. However, wtSV40 DNA was rapidly lost and replaced by a defective SV40 genome. The SV40 defective (E-SV40 alpha) contained a deletion of the late region, an intact early region, and paired with L-RFV clone H DNA to form a new hybrid virus. In human cells wtSV40 was also complemented by wtBKV DNA, but after two serial passages SV40 DNA disappeared. These findings indicate that SV40 late or capsid gene sequences, but not SV40 early sequences, generate a block to SV40 growth in human cells. When the SV40 late region is replaced by a RFV or a BKV late region, E-SV40 DNA propagates efficiently in human cells and in some cases more rapidly than wtBKV. Northern blot hybridization indicates that SV40 DNA is poorly transcribed in human cells when the SV40 late region is present.

Transformation of human cells by a polyomavirus containing complementing JCV and RFV genomes.

Molecularly cloned viral DNA from late RFV (L-RFV) and early JCV (E-JCV) were transfected into human fetal brain (HFB) cells and complementation was demonstrated. A new infectious virus (E-JCV/L-RFV) was produced. Infection resulted in partial transformation of HFB and human embryonic kidney cells. No transformation was observed with EL-JCV or wtJCV. The transformants contained T-antigen and had a lifespan similar to SV40-transformed human cells but failed to express some phenotypes of transformation. All transformants contained E-JCV viral DNA, usually both integrated and episomal. Although no L-RFV DNA was present in the transformants, L-RFV appears to play a role in the initiation of transformation.

Late coding region sequences required for competition by SV40 defectives.

SV40 defectives containing the complete early coding region (E-SV40) or the complete late region (L-SV40) were separately transfected into green monkey cells. They were analyzed for their ability to compete with wtSV40 (introduced by infection) or to undergo replication in the presence of constitutively produced SV40 T-antigen. L-SV40 competed very strongly. It appeared rapidly in infected cells, overgrowing wt genomes by at least 10:1. In addition, it slowed the growth of wt virus and reduced its ability to kill cells. L-SV40 DNA, as expected, replicated continuously in Cosl cells. E-SV40 genomes were poor competitors. They appeared slowly and by themselves did not overgrow wtSV40. When transfected into Cosl cells, E-SV40 genomes replicated efficiently for the first few days and disappeared within a week. Deletion or insertion mutations were introduced into a molecular clone of L-SV40, within the Vp1 gene or the Vp2 gene. All mutants were unable to form infectious virus in two different assays. The mutants were then assayed for competition against wtSV40 and for replication in Cosl cells. The Vp1 mutants competed very poorly with wt genomes and were rapidly lost from coinfected cells. These mutants, like E-SV40, replicated for only a few days in Cosl cells. In contrast, the Vp2 mutant competed with wtSV40 nearly as well as L-SV40. It also replicated continuously rather than transiently in Cosl cells. Next, we determined whether L-SV40 could effectively compete with other evolved SV40 defectives, not containing the late region but containing up to nine SV40 origin regions. We have shown that within five serial passages, L-SV40 became the predominant viral DNA species and the other defectives were lost. Although the Vp1 mutants and E-SV40 were weak competitors, they were shown to recombine with wtSV40 genomes to generate new L-SV40 genomes which again became the predominant species of viral DNA. These results demonstrate that L-SV40 is a potent competitor and that the Vp1 gene or a part of Vp1 plays an important role in this extraordinary competition. We suggest that the Vp1 gene functions to allow L-SV40 genomes to persist rather than generating a product which directly interferes with wtSV40 replication.

Complementation between SV40 and RFV defectives and acquisition of SV40 origins by late RFV genomes.

EL SV40 and RFV are variants of SV40 and BKV which contain bipartite or dual genomes. One molecule contains all the early viral sequences (E-SV40, E-RFV) and the other all the late viral sequences (L-SV40, L-RFV). Early and late genomes complement one another during productive infection. Experiments were designed to determine if E-genomes of one virus could complement L-genomes of another virus. If complementation did occur, intermolecular recombination events which lead to a more efficient infection or an altered host range might occur, and the sequences involved could than be identified. Two combinations were generated by direct transfection of BSC-1 green monkey cells. E-RFV and L-SV40 DNA complementation resulted in hybrid virus growth and cell killing. The hybrid demonstrated a narrow host range. Following serial passage, some E-RFV genomes contained SV40 origin region sequences but these recombinants did not overgrow prototype E-RFV genomes, even after many virus passages. In addition, no significant alterations in host range could be detected. Complementation between E-SV40 and L-RFV yielded a virus with a relatively wider host range. Virus growth and cell killing appeared very slowly at first. However, with each passage of E-SV40/L-RFV, cell killing occurred progressively more rapidly, until passage 7 when it became extensive in 7 days rather than 6-8 weeks. Infected cells contained 10-20 times more E-SV40 than L-RFV DNA during the first passage. However, by passage 7, both genomes were equally represented. During serial passage, L-RFV DNA acquired SV40 sequences from around the origin and the terminus of replication, such that recombinant (r) L-RFV genomes contained three SV40 origins [corrected] (including the 72-bp repeat) and 2 termini, and prototype L-RFV DNA was lost. E-SV40/rL-RFV demonstrated an altered host range propagating in some cell lines which did not support E-SV40/L-RFV growth. Both the host range change and the increased growth of rL-RFV genomes were shown to be at least partly caused by the acquisition of the SV40 sequences.

Isolation of a papovavirus with a bipartite genome containing unlinked SV40 and BKV sequences.

Wild-type (wt) BK virus was introduced into permissive BSC-1 cells along with either early or late defective SV40 genomes. The defectives contained all of the late (L-SV40) or all of the early (E-SV40) coding sequences. Persistently infected (PI) BSC-1 cultures were established and contained wt BKV DNA and E- or L-SV40 DNA in Hirt supernatants. Each of the BKV/SV40 combinations could be serially passed in BSC-1 cells. Also, DNase I digestion of virus stocks from BKV/E-SV40 infections did not eliminate E-SV40. This suggested that (1) E-SV40 genomes could be packaged in BKV capsids and (2) BKV T antigen acted to stimulate the growth of L-SV40 genomes. During continuous culture of PI BSC-1 cells containing BKV and L-SV40, wt BKV genomes were lost and replaced by a BKV defective. The BKV defective (E-BKV) contained a deletion in the late region, an intact early region, and a duplication of the origin. This combination represents a new papovavirus with a bipartite genome in which the early region is derived from BKV and the late region from SV40, and both are present in separate molecules. The BKV and SV40 defectives complement each other for infectivity. Infectious virus is formed with the E-BKV genomes packaged in SV40 capsids. It is hypothesized that this kind of recombination (reassortment) is a way in which papovaviruses may generate variation. The host range for the new BKV/SV40 is narrow. It propagates well in BSC-1 cells, relatively poorly in fetal human brain cells, and not at all in green monkey TC-7 or human embryonic kidney cells. However, it transforms fetal human brain cells at a frequency 25-50 times greater than wt BKV does.

High-performance liquid chromatographic mapping and structural characterization of neurophysin isoforms.

Both ion-exchange and reverse-phase HPLC protocols for micromapping of neurophysins have been examined and the structural relationships among the major isoforms identified in the maps have been characterized. Reverse-phase HPLC was found to be especially useful for obtaining fingerprints of the isoforms within each of the two major families of neurophysins, I (oxytocin-related) and II (vasopressin-related), for both bovine and human neurophysins from posterior pituitary sources. From fractionation of the bovine proteins on octylsilyl columns, at least four neurophysins I were identified, one of which corresponds to the intact sequence of 93 residues and three of which vary from the parent by various degrees of carboxyl-terminal truncation. For bovine neurophysin II, two isoforms were identified in the reverse-phase HPLC maps, both of 95 residues, which vary from one another by the residue, either Ile or Val, at position 89. Isoforms were also detected for human neurophysins, including a carboxyl-terminal truncated form of human neurophysin II. All of the major neurophysin isoforms and several of the minor forms were shown to be functionally active as expressed by their binding to peptide ligand affinity matrices. Reverse-phase HPLC mapping on the octylsilyl matrix allowed neurophysin fingerprinting of crude tissue extracts by providing a narrow "window" within which the neurophysins elute but many other polypeptides expected to be present are excluded. The reverse phase HPLC method provides a useful way to obtain isolated neurophysin isoforms for physicochemical characterizations now usually carried out with mixtures of isoforms obtained by ion-exchange chromatography. The method also has characteristics amenable both for high-sensitivity fingerprinting of neurophysin isoforms, from different species and anatomical sources, and as a prelude to microstructural and -functional characterization of the isoforms so isolated.

Proliferative myositis in the head and neck.

Benign fibrous lesions in the head and neck include lesions with a wide range of histologic features. Some of these conditions may be mistaken for malignant tumors, leading to unnecessary radical therapy. Proliferative myositis is one such lesion. A 49-year-old man had a one-week history of swelling over the left side of this mandible and upper neck. Most of the mass was removed and the diagnosis of proliferative myositis was confirmed by excisional biopsy. The patient was free of disease one year after surgery.

Pulmonary vascular response to endotoxin in normal and lymphocyte depleted sheep.

The cardiopulmonary effects of intravenously administered Escherichia coli endotoxin were studied in unanesthetized sheep. One group of animals was depleted of circulating T-lymphocytes while a non-depleted group served as control. T-lymphocyte depletion was accomplished by chronic thoracic duct drainage of lymph, removal of lymphocytes by continuous flow centrifugation and return of the cell free lymph intravenously. The T-lymphocyte depleted sheep demonstrated markedly obtunded increases in pulmonary arterial pressure and pulmonary vascular resistance following endotoxin when compared to the effects of the lipopolysaccharide in control animals. additionally, the lymphocyte depleted group showed a significant augmentation of myocardial contractility which occurred at the same time as marked systemic hypotension. This period of extreme hypotension following endotoxin is presumed to be accompanied by a reflex increase in the activity of the sympathetic nervous system. The control sheep, although equally hypotensive at this time, did not demonstrate a significant increase in myocardial contractility from the preendotoxin value. The results of these experiments indicate that T-lymphocytes may mediate some of the pathophysiological effects of bacterial endotoxin on the cardiovascular system.

Effect of nicotinic acid on vascular permeability after thermal trauma in the rat.

Nicotinic acid has been suggested to decrease plasma volume loss after thermal injury. However, conflicting data have recently appeared, in reports of laboratory measurements of major derangements in cardiovascular function after large third-degree thermal injury. We investigated the microvascular effect of nicotinic acid on water and albumin leakage after a small second-degree thermal burn in the rat. No effect of nicotinic acid on albumin leakage was observed at 1/2 hour, 3 hours, or 6 hours; a minimal but significant (p less than 0.05) decrease in water content of burned tissue was observed 1/2 hour postinjury. Our studies confirm in rats previous work with sheep and dogs demonstrating that nicotinic acid has slight, if any, effect on fluid and protein loss after thermal injury.

Cardiorespiratory effects of Pseudomonas and E. coli endotoxins in the awake dog.

A comparison of the cardiovascular and respiratory effects of Pseudomonas aeruginosa and Escherichia coli endotoxins was investigated in the unanesthetized dog. Animals were anesthetized with halothane for placement of cardiovascular catheters and then allowed to awaken prior to collection of control data and experimentation. One group of 12 animals was given E. coli endotoxin (5 mg/kg), another group of 13 received Pseudomonas endotoxin (8 mg/kg). Variables were collected for 6 h after endotoxin injection. A third group of 13 animals serving as sham animals received no endotoxin. When major cardiovascular variables, such as arterial blood pressure, cardiac output, right atrial pressure, and left ventricular pressure and dP/dt were monitored, it was seen that the basic patterns of response to endotoxins were quite similar, with differences between groups being primarily quantitative. Analysis of respiratory data showed that animals receiving Pseudomonas developed an earlier respiratory response. Nevertheless, blood gas data were similar in the two groups.

Differential response to cytochalasin B among cells transformed by DNA and RNA tumor viruses.

Mouse, hamster, rat, human, and chick cells were transformed by RNA and DNA tumor viruses: simian virus 40, adenovirus type 7, Kirsten mouse sarcoma virus (Ki-MuSV), Moloney mouse sarcoma virus, and Rous sarcoma virus. All cultures of transformed cells grew to high concentration densities. Normal and transformed cells were treated with cytochalasin B (CB) at concentrations preventing cytoplasmic cleavage. Cells altered by DNA tumor viruses responded to CB with numerous nuclear divisions resulting in highly multinucleated cells. All but one line of cells transformed by RNA tumor viruses responded to CB with usually only one and occasionally two nuclear divisions. Only binucleated cells were formed. One clone of CB-treated BALB/c mouse embryo fibroblasts transformed by Ki-MuSV showed numerous cells with four and five nuclei. HOWEVER, IN CONTRASt to cells transformed by DNA viruses, few cells had seven or more nuclei. These results suggest that, in the presence of CB, cells transformed by DNA tumor viruses show uncontrolled nuclear division, whereas cells tranformed by RNA tumor viruses show controlled nuclear division.