Health-Related Quality-of-Life Outcomes: Comparing Patients With Aneurysmal and Nonaneurysmal Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) is divided into two major types (aneurysmal [ASAH] and nonaneurysmal [NASAH]) because, in approximately 15% of the patients who experience SAH, no source of hemorrhage can be identified. Anecdotal evidence and contradictory research suggest that patients with NASAH experience some of the same health-related quality of life (HRQOL) issues as patients with ASAH. This quantitative survey design study compared 1-3 years after hemorrhage the HRQOL in patients who had experienced an NASAH with those who had experienced an ASAH. This is the first U.S. study to specifically investigate HRQOL in NASAH and the second to compare HRQOL outcomes between patients with ASAH and NASAH. These study results corroborate those of the first-that the two groups are much more similar than different. It confirms that the impact on employment for both hemorrhage groups is significant, and it also finds an even greater inability to return to work for the patients with NASAH. Physical symptom complaints were more common in the group with NASAH, whereas the group with ASAH experienced more emotional symptoms. Both groups had low levels of posttraumatic stress disorder (PTSD), with those levels not differing significantly between groups. However, PTSD and social support were shown to impact HRQOL for both groups. The authors recommend that clinicians assess all patients with SAH for PTSD and institute treatment early. This may include offering psychological services or social work early in the hospital course. Further research and policy changes are needed to assist in interventions that improve vocational reintegration after SAH. Patients with NASAH should no longer be described as having experienced a "benign hemorrhage." They have had a life-changing hemorrhage that may forever change their lives and impact their HRQOL.

Development of cell lines from the cactophagous insect: Cactoblastis cactorum (Lepidoptera: Pyralidae) and their susceptibility to three baculoviruses.

The unintentional introduction of the cactus moth, Cactoblastis cactorum, a successful biological control agent formerly employed in the control of invasive prickly pear cactus species (Opuntia spp.) in Australia, Hawaii, South Africa, and various Caribbean islands, has posed great concern as to the possible threat to native, endangered species of cactus in the southeastern USA as well as with the potential to cause a major infestation of commercial and agricultural cactus crops in Mexico. A number of control measures have been investigated with varying degrees of success including, field exploration for cactus moth-specific parasitoids, insecticides, fungal, bacterial, and nematode agents. Current tactics used by the USA-Mexico binational program to eradicate cactus moth from Mexico and mitigate its westward movement in the USA include host plant removal, the manual removal and destruction of egg sticks and infected cacti stems, and the Sterile Insect Technique. One other approach not taken until now is the development of a cactus moth cell line as a tool to facilitate the investigation of baculoviruses as an alternative biocontrol method for the cactus moth. Consequently, we established C. cactorum cell lines derived from adult ovarian tissue designated as BCIRL-Cc-AM and BCIRL-Cc-JG. The mean cell population doubling time was 204.3 and 112 h for BCIRL-Cc-AM and BCIRL-Cc-JG, respectively, with weekly medium change, while the doubling time was 176.6 and 192.6 h for BCIRL-Cc-AM and BCIRL-Cc-JG, respectively, with a daily change of medium. In addition, the daily versus weekly change in medium was reflected in the percentage viability with both cell lines showing higher levels with a daily medium change. Of the three baculoviruses tested, only the recombinant AcMNPV-hsp70Red and GmMNPV at a multiplicity of infection (MOI) of 1.0 were able to demonstrate significant production of extracellular virus (ECV) in each of the cell lines, whereas both cell lines were refractive to an HzSNPV challenge at an MOI of 10. In this study, we have demonstrated both the successful development of a C. cactorum cell line and its ability to support a complete baculovirus infection. The potential is also there to pursue further investigations to determine the susceptibility of the cactus moth cell line to other viruses. Additionally, the availability of a cactus moth cell line will facilitate the analysis of viruses prior to using the more expensive bioassay test. Finally, it is hoped with the knowledge presented here that baculoviruses may also be considered as an alternative biocontrol method for the cactus moth.

Baculovirus infection influences host protein expression in two established insect cell lines.

We identified host proteins that changed in response to host cell susceptibility to baculovirus infection. We used three baculovirus-host cell systems utilizing two cell lines derived from pupal ovaries, Hz-AM1 (from Helicoverpa zea) and Hv-AM1 (from Heliothis virescens). Hv-AM1 cells are permissive to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and semi-permissive to H. zea single nucleopolyhedrovirus (HzSNPV). Hz-AM1 cells are non-permissive to AcMNPV. We challenged each cell line with baculovirus infection and after 24h determined protein identities by MALDI TOF/TOF mass spectrometry. For Hv-AM1 cells, 21 proteins were identified, and for Hz-AM1 cells, 19 proteins were newly identified (with 8 others having been previously identified). In the permissive relationship, 18 of the proteins changed in expression by 70% or more in AcMNPV infected Hv-AM1 cells as compared with non-infected controls; 12 were significantly decreased and 6 cellular proteins were significantly increased. We also identified 3 virus-specific proteins. In the semi-permissive infections, eight proteins decreased by 2-fold or more. Non-permissive interactions did not lead to substantial changes in host cell protein expression. We hypothesize that some of these proteins act in determining host cell specificity for baculoviruses.

Prostaglandins A1 and E1 influence gene expression in an established insect cell line (BCIRL-HzAM1 cells).

Prostaglandins (PGs) and other eicosanoids exert important physiological actions in insects and other invertebrates, including influencing ion transport and mediating cellular immune defense functions. Although these actions are very well documented, we have no information on the mechanisms of PGs actions in insect cells. Here we report on the outcomes of experiments designed to test our hypothesis that PGs modulate gene expression in an insect cell line established from pupal ovarian tissue of the moth Helicoverpa zea (BCIRL-HzAM1 cells). We treated cells with either PGA(1) or PGE(1) for 12 or 24h then analyzed cell lysates by 2-D electrophoresis. Analysis of the gels by densitometry revealed substantial changes in protein expression in some of the protein spots we analyzed. These spots were processed for mass spectrometric analysis by MALDI TOF/TOF, which yielded in silico protein identities for all 34 spots. The apparent changes in three of the proteins were confirmed by semi-quantative PCR, showing that the changes in mRNA expression were reflected in changes in protein expression. The 34 proteins were sorted into six categories, protein actions, lipid metabolism, signal transduction, protection, cell functions and metabolism. The findings support the hypothesis that one mechanism of PG action in insect cells is the modulation of gene expression.

Isolation and characterization of a baculovirus associated with the insect parasitoid wasp, Cotesia marginiventris, or its host, Trichoplusia ni.

A multiple nucleopolyhedrovirus (MNPV) was isolated from Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) larvae that had been stung by the parasitoid Cotesia marginiventris (Cresson) (Hymenoptera: Braconidae). The wild type virus was plaque purified by infecting a Heliothis subflexa (BCIRL- HsAM1) cell line and isolating several clones. The mean estimated genomic size of this virus based on PstI, BstEII, StyI, HindIII restriction profiles was estimated to be 106 +/- 2.5 kbp (mean+/-SE). A clone designated as TnMNPV/CmBCL9 was used in bioassays against several lepidopteran pests and in comparative studies with the baculoviruses AcMNPV, AgMNPV, AfMNPV, PxMNPV and HzSNPV of Autographa califomica, Anticarsia gemmatalis, Anagrapha falcifera, Plutella xylostella, and Helicoverpa zea, respectively. Infectivity studies showed that TnMNPV/CmBCL9 was highly infectious for Heliothis subflexa and T. ni, with an LC(50) value 0.07 occlusion bodies/mm(2) in both species and also infectious for H. zea and Heliothis virescens with LC(50) values of 0.22 and 0.27 occlusion bodies/mm(2), respectively. Restriction endonuclease analysis of the isolate and selected baculoviruses revealed profiles that were very similar to AfMNPV but different from the restriction endonuclease profiles of the other baculoviruses. Hybridization studies suggest that the TnMNPV/CmBCL9 was closely related to AfMNPV and AcMNPV-HPP. Further support for this comes from a phylogenetic analysis employing a split-graphs network, comparing the polh, egt, and p10 genes from TnMNPV/CmBCL9 with those from other baculoviruses and suggests that this virus is closely related to the AcMNPV variants, AfMNPV and RoMNPV of Rachiplusia ou.

In vitro host range of the Hz-1 nonoccluded virus in insect cell lines.

A total of 13 insect cell lines spanning 4 orders (Lepidoptera, Coleoptera, Diptera, and Homoptera) were tested for their ability to replicate the nonoccluded virus Hz-1. Only the Lepidopteran cell lines supported replication of the virus with TN-CL1 and BCIRL-HZ-AM1 producing the highest titers of 2.4 x 10(8) tissue culture infective dose (TCID)50/ml and 2.0 x 10(8) TCID50/ml, respectively. A codling moth cell line (CP-169) was the only Lepidopteran cell line that did not replicate the virus and transfection of this cell line with Hz-1 DNA failed to replicate the virus. Also, transfection with DNA from a recombinant baculovirus carrying the red fluorescent protein gene (AcMNPVhsp70 Red) was not expressed in CP-169 cells. The replication cycle of Hz-1 in BCIRL-HZ-AM1 cells showed that this virus replicated rapidly starting at 16 h postinoculation (p.i.) and reaching a peak titer of 1.0 x 10(8) TCID50/ml 56 h postinoculation. Hz-1 when compared with several other baculoviruses has the widest in vitro host spectrum.

Cross-species investigation of Helicoverpa armigera microsatellites as potential markers for other related species in the Helicoverpa--Heliothis complex.

Primers previously designed to amplify microsatellite DNA markers in the Old World bollworm, Helicoverpa armigera, larvae were tested in three closely related species: the corn earworm, Helicoverpa zea, tobacco budworm, Heliothis virescens, and Heliothis subflexa. Of the fourteen loci surveyed, only four loci (HaB60, HaC14, HaC87, HarSSR1) consistently demonstrated scorable single-copy microsatellite bands. Of these four, length polymorphism was identified only in the HaB60 marker (160 bp, 140 bp) of the H. virescens and H. subflexa sampled laboratory populations. Partial DNA sequences of all the identified single-copy microsatellites are presented as well as alignments to their respective H. armigera microsatellite.

AcMNPV in permissive, semipermissive, and nonpermissive cell lines from Arthropoda.

Insect cell lines from Arthropoda represented by Lepidoptera, Coleoptera, Diptera, and Homoptera were evaluated for their ability to support replication of AcMNPV. In addition, some of the cell lines that were refractive to AcMNPV were tested with AcMNPV hsp70 Red, a recombinant carrying the red fluorescent protein (RFP) gene, for their ability to express this protein after inoculation. Of the 10 lepidopteran cell lines tested, only three cell lines from Helicoverpa zea (BCIRL-HZ-AM1), Lymantria dispar (IPLB-LD 65), and Cydia pomonella (CP-169) failed to support detectable viral replication as measured by tissue culture infectious dose 50 (TCID50) assay. Heliothis virescens (BCIRL-HV-AM1) produced the highest viral titer of 2.3 +/- 0.1 x 10(7) TCID50/ml followed by Heliothis subflexa (BCIRL-HS-AM1) at 4.7 +/- 0.1 x 10(6) TCID50/ml and Spodoptera frugiperda (IPLB-SF21) at 4.1 +/- 0.1 x 10(6) TCID50/ml. None of the coleopteran, dipteran, or homopteran cell lines supported AcMNPV replication. However, when studies were performed using AcMNPV hsp70 Red, the dipteran cell lines Aedes aegypti (ATC-10) and Drosophila melanogaster (line 2), both expressed the RFP as well as the refractive lepidopteran cell lines from H. zea and L. dispar. No RFP expression was observed in any of the coleopteran or homopteran cell lines. Cell lines refractive to AcMNPV did not appear to be adversely affected by the virus, as judged by their ability to multiply, nor was there any indication of induced apoptosis, as assessed by deoxyribonucleic acid fragmentation profiles or cell blebbing, or both.

Development and partial characterization of heliothine cell lines from embryonic and differentiated tissues.

The goal of this study was to generate cell lines from a variety of insect tissues that could be useful for developing in vitro assays with tissue-specific properties. In this article, we describe the establishment of new cell cultures from differentiated (primarily neural) and undifferentiated tissues (primarily embryonic) and their initial characterization. Cell lines were established from the following tissues of the budworm, Heliothis virescens, and the bollworm, Helicoverpa zea: larval ventral nerve cords (4 lines), larval midguts (1 line), adult ovaries (1 line), and embryonic tissues (11 lines). Cell lines were primarily characterized by morphological examination and polymerase chain reaction (PCR) (both deoxyribonucleic acid amplification fingerprinting and inter-simple sequence repeats PCR).

Providence virus: a new member of the Tetraviridae that infects cultured insect cells.

We identified a new member of the Tetraviridae, Providence virus (PrV), persistently infecting a midgut cell line derived from the corn earworm (Helicoverpa zea). Virus purified from these cells also productively infected a H. zea fat body cell line, and a cell line from whole embryos of the beet armyworm, Spodoptera exigua. PrV is thus the first tetravirus shown to replicate in cell culture. PrV virions are isometric particles composed of two structural proteins (60 and 7.4 kDa) that encapsidate both the genomic (6.4 kb) and the subgenomic (2.5 kb) RNAs. The monopartite organization of the PrV genome resembles that of Nudaurelia beta virus and Thosea asigna virus, members of the genus Betatetravirus. The predicted sequence of the PrV structural proteins demonstrates homology to tetraviruses in both genera. The infectivity of PrV for cultured cells uniquely permitted examination of tetravirus RNA and protein synthesis during synchronous infection. The discovery of PrV greatly facilitates studies of tetravirus molecular biology.

Application of inter-simple sequence repeats to insect cell lines: identification at the clonal and tissue-specific level.

Inter-simple sequence repeat (ISSR) primers designed to anneal to microsatellites were used to obtain deoxyribonucleic acid (DNA) fingerprint profiles to distinguish among 16 established insect cell lines derived from an assortment of lepidopteran, dipteran, and coleopteran species. Three different levels of cell line comparison were made: (1) between parents and their clones, (2) among cell lines derived from different tissues from the same species, and (3) among cell lines derived from different insect species. Of the 16 repeat oligonucleotide primers used in this study, nine primers generated several unique markers to distinguish between parental cell lines and their clones. Four of the 16 primers also generated DNA profiles with a number of unique bands, enabling the distinction among cell lines derived from specific tissues from the same species. In addition, ISSR-generated DNA profiles provided the greatest number of unique markers to distinguish easily among insect cell lines derived from different species.

Insect cells and their potential as stabilization barriers for DNA of multiple and single nucleopolyhedroviruses against ultraviolet-B-simulated sunlight inactivation.

A cell line from Trichoplusia ni (TN-CL1) infected with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV-HPP) and a cell line from Helicoverpa zea (BCIRL-HZ-AM1) infected with the Helicoverpa zea single nucleopolyhedrovirus (HzSNPV/BrCL2) were subjected to ultraviolet-B (UV-B) irradiation at a predetermined level of exposure that would inactivate greater than 95% of the virus suspended in the liquid. The working hypothesis was that the homologous insect cells would utilize their inherent deoxyribonucleic acid (DNA) repair mechanism(s) to prevent, repair, or at least mitigate the damaging effects of UV-B light on viral DNA synthesis. We attempted to determine this by using infected cells that were subjected to UV-B irradiation at different postinoculation periods under two experimental conditions of exposure: (1) shielded, and (2) nonshielded. Of the two cell lines infected with their respective homologous viruses, the virus from TN-CL1 cells was the least sensitive to UV-B light because the extracellular virus (ECV) and occlusion body (OB) levels of virus-infected TN-CL1 cells were higher than those of the virus-infected BCIRL-HZ-AM1 cells. Production of ECV and OB from both cell lines was lower in the exposed, nonshielded treatment than in the exposed, shielded treatment. However, AcMNPV-HPP was produced in enough quantity to indicate that TN-CL1 might impart a level of protection to the virus against UV light.

Effect of neonatal thymectomy and antithymocytic serum on susceptibility of rats to mycobacterium leprae infection

Neonatal thymectomy and neonatal thymectomy plus ATS resulted in a merked increase in susceptibility of both Buffalo and Lewis rats to infection with M. leprae. In Buffalo rats, increase in susceptibility was limited to footpad infection. In Lewis rats, this was extended to include testis infection where the organisms in thymectomized-ATS-treated rats were found to be still in the logarithmic phase of growth 15 months after inoculation.