Measles virus: Evolution of a persistent infection in BGM cells

Summary An African green monkey kidney cell line (BGM) persistently infected with measles virus (BGM/Hallé cells) has been studied during 3 years in culture. The early cell passages were characterized by slow growing cultures producing high yields of infections virus (10⁶–10⁷ PFU/ml). These cells were gradually replaced by a population of cells multiplying at a similar rate as non-infected cells. During this evolution, the virus released from the cells changed from a large plaque variant, to a small plaque and eventually unlysed foci. Despite 95 per cent of the cells being infected, the virus yield fell to the limits of detection.[³⁵S]-methionine labelling of BGM/Hallé cultures at the 20th and 140th passage showed that in both cases all the measles virus structural proteins were synthesized. There were no changes in the apparent molecular sizes of the viral proteins during passage.Cell surface [¹²⁵I]-labelling of BGM/Hallé cells indicates that viral envelope antigens are inserted into the membrane despite the diminution in virus yield. Several cell proteins not labelled in non-infected BGM cells are also labelled. These proteins could also be [¹²⁵I]-labelled in clones of BGM/Hallé cells with had been cured of virus.Chase experiments of [¹²⁵I]-pre-labelled BGM/Hallé cultures showed the radio-labelled antigen to be incorporated into virus particles. Non-infections virus particles released from the cells contained the same polypeptides as those released from a lytic infection.With 7 Figures     

Papillary cystic tumours of the pancreas: An analysis by nuclear morphometry

Papillary cystic tumour (PCT) is a rare, low-grade malignant pancreatic neoplasm, in which the histological criteria for malignancy are still uncertain. We performed a histological examination of 3 metastasizing PCTs, while comparing them with 18 non-metastasizing PCTs, using a computed image analyser. The mean maximum nuclear diameter, the mean standard deviation (SD) of the nuclear diameter, the mean nuclear area and the nuclear-nonnuclear (N/NN) ratio obtained by the image analyser of the metastasizing PCTs (7.23 m, 2.21 m, 30.45 m², 36.41%) were all significantly larger than those of the non-metastasizing PCT (6.34 m, 1.59 m, 23.66 m², 23.74%;P<0.005,P< 0.005,P<0.005,P<0.001 respectively). However, there were no statistical differences in either the nuclear ellipsoidity or nuclear regularity. These results suggested that nuclear morphometry might be a useful parameter to define metastatic potential, in addition to histological variables such as venous invasion, nuclear grade and mitotic rate.     

Reliability and Validity Evidence of Multiple Balance Assessments in Athletes With a Concussion

Context:

An estimated 300 000 sport-related concussion injuries occur in the United States annually. Approximately 30% of individuals with concussions experience balance disturbances. Common methods of balance assessment include the Clinical Test of Sensory Organization and Balance (CTSIB), the Sensory Organization Test (SOT), the Balance Error Scoring System (BESS), and the Romberg test; however, the National Collegiate Athletic Association recommended the Wii Fit as an alternative measure of balance in athletes with a concussion. A central concern regarding the implementation of the Wii Fit is whether it is reliable and valid for measuring balance disturbance in athletes with concussion.

Objective:

To examine the reliability and validity evidence for the CTSIB, SOT, BESS, Romberg test, and Wii Fit for detecting balance disturbance in athletes with a concussion.

Data Sources:

Literature considered for review included publications with reliability and validity data for the assessments of balance (CTSIB, SOT, BESS, Romberg test, and Wii Fit) from PubMed, PsycINFO, and CINAHL.

Data Extraction:

We identified 63 relevant articles for consideration in the review. Of the 63 articles, 28 were considered appropriate for inclusion and 35 were excluded.

Data Synthesis:

No current reliability or validity information supports the use of the CTSIB, SOT, Romberg test, or Wii Fit for balance assessment in athletes with a concussion. The BESS demonstrated moderate to high reliability (interclass correlation coefficient = 0.87) and low to moderate validity (sensitivity = 34%, specificity = 87%). However, the Romberg test and Wii Fit have been shown to be reliable tools in the assessment of balance in Parkinson patients.

Conclusions:

The BESS can evaluate balance problems after a concussion. However, it lacks the ability to detect balance problems after the third day of recovery. Further investigation is needed to establish the use of the CTSIB, SOT, Romberg test, and Wii Fit for assessing balance in athletes with concussions.Key Words: Clinical Test of Sensory Organization and Balance, Sensory Organization Test, Balance Error Scoring System, Romberg test, Wii Fit, sensitivity, specificity

Key Points

• No current reliability or validity data exist to support the use of the Wii Fit to diagnose balance impairments in athletes with concussions.
• The Balance Error Scoring System has moderate to high reliability and low to moderate validity evidence to support its use in diagnosing balance impairment in athletes with concussions.

An estimated 300 000 sport-related concussion injuries occur in the United States per year.¹ The rate of sport-related concussion is anticipated to rise as sports involvement increases at the collegiate and high school levels.² Concussion has been defined by the Third International Conference on Concussion in Sport as a complex pathophysiologic process affecting the brain induced by traumatic biomechanical forces.^3,4 Potential concussion symptoms include confusion, loss of memory, loss of consciousness, loss of spatial and temporal awareness, headaches, migraines, speech impairment, dizziness, nausea, balance disturbances, oculomotor control reduction, vision impairment, speech reduction, gait unsteadiness, and poor coordination.³ Because of the variable symptoms experienced by affected athletes and the multiple mechanisms of injury that can contribute to a concussive injury, no 2 concussions are perfectly similar.³ This hinders accurate assessment and management of sport-related concussion.No single tool can fully quantify a concussion injury.⁵ Yet measures that focus on the most prevalent symptoms experienced by individuals after a concussion injury could help in diagnosis. The most common symptoms are headaches, dizziness, and balance problems.^5,6 Of the 3 top symptoms, dizziness and balance dysfunction are related. Thus, examining balance dysfunction after a concussion could provide a quantitative, objective measure of the injury.Of individuals suffering from a sport-related concussion, 30% report balance dysfunction and 75.6% report dizziness as a debilitating symptom.^7,8 Balance disturbances have been noted to return to normal within 72 hours; however, prolonged damage may last more than 7 days beyond the initial injury.^6,9 Balance disturbance is the inability to stand with an upright posture without deviating outside the limits of the base of support.⁶ After concussion, problems with the vestibular system are considered most likely to be responsible for the individual''s inability to maintain balance.⁶ Two candidate mechanisms have been proposed to underlie diminished vestibular function with concussion: (1) damage to peripheral receptors or (2) inhibited sensory integration in response to structural damage of the central processing structures.⁶ Vestibular dysfunction is a considerable detriment to activities of daily living and athletics. A balance disturbance could place the athlete at greater risk for additional injury through falls or collisions. Therefore, we need to accurately assess balance in athletes with concussions.     

Direct induction of tetraploids or homozygous diploids in the industrial yeast Saccharomyces cerevisiae by hydrostatic pressure

Summary Hydrostatic pressure and a dye plate method were used to investigate the direct induction of tetraploids or homozygous diploids from the industrial diploid or haploid yeast Saccharomyces cerevisiae. Above 200 MPa, hydrostatic pressure greatly inactivated the strains HF399s1 ( haploid), P-540 (a/ diploid), and P-544 (a/ diploid). At the same time, when pressure-treated cells of these strains were spread on a dye plate, some of the visible colonies were stained red/blue or dark blue (variant colonies); the rest stained violet, similar to colonies originating from diploid cells or haploid cells that were not pressure-treated. In addition, above 100 MPa, the formation of variant colonies increased with increasing pressure, and maximized (1x10^-1) at 200 and 250 MPa, respectively. The size of almost all variant cells from P-544, P-540, and HF399s1 was visibly increased compared with that of untreated cells and the measured cellular DNA content of P-540 and HF399s1 was double that of untreated cells. Furthermore, based on random spore analysis and mass-matings, induced variants in the diploid strains were found to be tetraploid with an a/a// genotype at the mating-type locus or, in the haploid strains, homozygous diploid with an / genotype. From these results we conclude that pressure treatment in combination with a dye plate is a useful method for strain improvement by direct induction of tetraploids or homozygous diploids from industrial strains whether diploid or haploids.     

Evidence for the type of nucleic acid in African swine fever virus

Summary The growth of African swine fever virus (ASFV) in pig kidney cells (PK-15) was inhibited by 5-iodo- and 5-bromo-2-deoxyuridine (IUDR and BUDR), whereas the fluoro-compound was inactive in this respect. In another cell line of the same origin (PK-C) virus inhibition occurred with as little as 1g/ml. of BUDR. The inhibitory effect was reduced when the compound was added more than 6 hours after infection, i.e. about 4 hours preceding the first appearance of progeny virus and was completely reversed in the presence of excess thymidine. Cultures in which virus multiplication was completely inhibited showed no cytopathic effects. Actinomycin-D also suppressed the growth and cytopathogenicity of ASFV in PK-C cells, the minimal inhibitory concentration being 0.2g/ml.; the antibiotic-sensitive phase of virus multiplication was 2–6 hours after infection.The growth of pseudorabies virus was inhibited in parallel with that of ASFV but the multiplication of foot-and-mouth disease virus in PK-C cells was not affected by these compounds.When ASFV was grown in the presence of¹⁴C-thymidine, followed by centrifugal concentration, enzymatic treatment and fractionation in sucrose density gradients, a peak of radioactivity coinciding with that of viral infectivity was obtained. No such peak occurred when the virus was grown in the presence of¹⁴C-uridine.It is concluded that ASFV is a DNA virus and should no longer be considered as a possible myxovirus.     

Persistence of Sindbis virus in BHK-21 cell cultures

Summary If BHK-21 cell monolayer cultures are inoculated with Sindbis virus individual surviving cells can be observed, which are capable to multiply thereby forming small colonies in which the virus persists. The virus causes periodical cell destructions usually starting in the centers of the colonies. Increasing virus titers are observed during the phases of cell destruction and decreasing titers during the phases of cell growth. It takes several weeks for the slow growing colonies to form confluent cell sheets. Those cultures can be trypsinized and grown in continuous passages. The above mentioned periodical cell destructions then do no longer occur because a more stable equilibrium between BHK-21 cells and Sindbis virus has been established. About 6% of the cells are virus producers as evidenced by infective center tests. The supernatant contains approximately 10⁴ to 10⁵ PFU per 0,1 ml. The behaviour of the cells is changed concerning plaque formation by Sindbis virus if the persistent infection is eliminated by addition of immune serum. Cell morphology, however, remains unchanged. Virus from persistently infected cells differs also from the original virus: it produces smaller plaques in BHK-21 cell and chick embryo fibroblast cultures.Partly presented at 3. Arbeitstagung der Deutschen Gesellschaft für Hygiene und Mikrobiologie, Mainz, October 8–10, 1970.     

Parvovirus (feline panleucopaenia virus) plaque formation

Summary A plaque assay was developed for feline parvovirus (FPV; feline panleucopaenia virus) in a feline embryo (FEmb) cell line. Higher numbers and larger diameter plaques were obtained with a) seeding rates of 0.7 × 10⁵ and 1.5×10⁵ cells cf. 3×10⁵ and 6×10⁵ cells/well of 35 mm diameter, b) synchronised cells infected at the G₁—S interface cf. nonsynchronised cells and c) 5 to 6 days incubation post inoculation.The plaque assay was standardised by using serum deprivation for 24 hours to synchronize cells, a seeding rate of 1.5×10⁵ cells/35 mm diameter well, inoculation of virus 16 hours post seeding followed by 5 days incubation. The standardised assay gave consistent, reproducible results. A dose-response curve using the assay showed a linear, 45° slope, relationship between plaque forming units and virus dilution which further verified the sensitivity and reliability of the assay. Plaques produced by wild type and plaque purified virus were inavriably non uniform in diameter; diameter of plaques in fact followed a normal frequency distribution under standard assay conditions.With 3 Figures     

Properties of virus associated with subacute myelo-optico-neuropathy

Summary The virus associated with subacute myelo-optico-neuropathy (S.M.O.N.) produced weak and incomplete cytopathic effect (CPE) in BAT-6 cells but not in other cells so far tested. However, the virus multiplied in human diploid cells without CPE. The virus passed a membrane filter of 220 nm average pore-size but not one of 100 nm. The virus was sensitive to ether and 5-fluoro-2-deoxyuridine, and was inactivated by ultraviolet irradiation. Furthermore, the virus was considered to contain DNA since it was labelled by³H-thymidine but not by uridine. The buoyant density of the virus was 1.21 to 1.22 g/ml in CsCl. This agent seems to be a new neuropathic slow virus.     

Correlation of virus replication,cytokine (TNF-α and IL-1) producing cells,neuronal necrosis and inflammation after intranasal infection of mice with herpes simplex virus strains of different virulence

Summary The number of TNF- and IL-1 producing cells was investigated during the acute replication phase of herpes simplex virus (HSV) in trigeminal ganglia after intranasal infection with strains of different virulence. The highly virulent strain WAL replicated strongly and induced many cytokine producing cells early in the ganglia. The low virulent strain HFEM replicated less, only few cytokine producing cells were detected late. The thymidine-kinase negative (TK^–) virus 1301 did not replicate but produced some lymphocytic inflammation. The higher the virulence of strains of HSV-1 or -2 was, the stronger was the extent of histopathological lesions; moreover, a dissociation in time between replication and cellular reaction (granulocytic and lymphocytic) could be observed after infection with strains HFEM and TK^– virus 1301. CD4 and CD8 positive cells could be detected mainly at the rim of necrotic areas, TNF- and IL-1 producing cells, however, were scattered throughout the ganglia.     

The effect of DEAE-Dextran on the infectivity of a feline calicivirus and its RNA

Summary The polycation DEAE-Dextran increases the plating efficiency of an intact feline calicivirus in primary kitten kidney cells by up to 10 times. Both pretreated virus and pretreated cells show a similar dose response curve with a peak at 250 g/ml DEAE-Dextran. Above this concentration, the polycation appeared toxic to the cell monolayers. Pretreated washed cells showed an increased plaque count of intact virus but this was only in the order of a two fold increase.Low titres of infectious RNA could be assayed from the virus. No infectious RNA could be detected in the absence of DEAE-Dextran and titres of RNA were 10^–6 of the intact virus.     

Association between serum-soluble CD8 levels and parameters of immune activation in patients with human immunodeficiency virus infection

Summary We compared the serum concentrations of soluble CD8 with the immune activation markers neopterin, interferon-, tumour necrosis factor-, soluble CD4, and with CD34⁺ and CD38⁺ T-cell counts in patients with human immunodeficiency virus (HIV) infection. The majority of patients had increased concentrations of soluble CD8, interferon- and neopterin, and various significant correlations existed between them. Our results support the view that enhanced soluble CD8 levels indicate activated CD8⁺ T cells in patients with HIV infection.Abbreviations sCD8 serum-soluble CD8 - sCD4 serum-soluble CD4 - IFN- interferon- - TNF =tumour necrosis factor- - HIV human immunodeficiency virus - AIDS acquired immunodeficiency virus     

Seasonal and Pandemic Human Influenza Viruses Attach Better to Human Upper Respiratory Tract Epithelium than Avian Influenza Viruses

Influenza viruses vary markedly in their efficiency of human-to-human transmission. This variation has been speculated to be determined in part by the tropism of influenza virus for the human upper respiratory tract. To study this tropism, we determined the pattern of virus attachment by virus histochemistry of three human and three avian influenza viruses in human nasal septum, conchae, nasopharynx, paranasal sinuses, and larynx. We found that the human influenza viruses—two seasonal influenza viruses and pandemic H1N1 virus—attached abundantly to ciliated epithelial cells and goblet cells throughout the upper respiratory tract. In contrast, the avian influenza viruses, including the highly pathogenic H5N1 virus, attached only rarely to epithelial cells or goblet cells. Both human and avian viruses attached occasionally to cells of the submucosal glands. The pattern of virus attachment was similar among the different sites of the human upper respiratory tract for each virus tested. We conclude that influenza viruses that are transmitted efficiently among humans attach abundantly to human upper respiratory tract, whereas inefficiently transmitted influenza viruses attach rarely. These results suggest that the ability of an influenza virus to attach to human upper respiratory tract is a critical factor for efficient transmission in the human population.Influenza is an important cause of morbidity and mortality in humans during seasonal, pandemic, and zoonotic outbreaks. Seasonal influenza is estimated to cause 250,000 to 500,000 deaths per year worldwide. Pandemic influenza viruses of the previous century resulted in an estimated 1 to 4 million deaths for the 1957 H2N2 (Asian flu) and the 1968 H3N2 (Hong Kong flu) influenza pandemics, and 20 to 50 million deaths for the 1918 H1N1 (Spanish flu) influenza pandemic.^1,2 The first influenza pandemic of the 21st century, the currently ongoing new H1N1 virus outbreak (Mexican flu), has caused at least 3486 deaths as of September 13, 2009 (http://www.who.int/csr/don/2009_09_18/en/index.html). The zoonotic highly pathogenic avian influenza virus (HPAIV) H5N1, which is causing an ongoing outbreak in poultry, only occasionally infects humans, but has a high mortality rate, with 262 deaths out of 400+ confirmed infections as of August 2009 (http://www.who.int/csr/disease/avian_influenza/country/cases_table_2009_08_11/en/index.html).The pandemic potential of an influenza virus depends largely on its efficiency of human-to-human transmission. Human influenza viruses, including seasonal H1N1 and H3N2 viruses, and the pandemic H1N1 virus, are transmitted efficiently.³ In contrast, the zoonotic HPAIV H5N1 is only rarely transmitted from human to human.⁴ However, the factors determining efficient virus transmission among humans are poorly understood.Tropism of influenza virus for the human upper respiratory tract (URT) has been speculated to be an important determinant for the efficiency of virus transmission, based both on receptor distribution and virus replication studies.^5,6 Based on lectin histochemistry, the human URT has abundant receptors for human influenza viruses, which are efficiently transmitted.^5,7 This fits with the ability for human influenza viruses to replicate in human URT tissues based on in vivo,⁸ ex vivo,⁷ and in vitro studies.^9,10,11 In contrast, the human URT has only limited receptors for avian influenza viruses.^5,7 This fits with the absence or rarity of HPAIV H5N1 transmission among humans.⁴ However, it is discordant with a study of Nicholls and others, who showed that HPAIV H5N1 can replicate in URT tissues. They explained this discordance by suggesting that HPAIV H5N1 attached to receptors not detected by the lectins used. Therefore, there is currently no consensus on the tropism of HPAIV H5N1 for the human URT. In addition, the studies to date have not studied the human URT systematically, and it is not known what the tropism of the new H1N1 virus is for the human URT.To address the question whether URT tropism of influenza viruses is linked to efficient transmission, we determined the pattern of attachment of selected influenza viruses in the human URT: human influenza viruses, including seasonal H1N1 and H3N2 viruses and pandemic H1N1 virus, which are transmitted efficiently, and avian influenza viruses, including a HPAIV H5N1, isolated from a fatal human case, which is not transmitted efficiently among humans. We measured the pattern of virus attachment by use of virus histochemistry instead of lectin histochemistry.⁶ Virus histochemistry measures the attachment of influenza virus to its host cell directly. Therefore, any receptors other than SA-α-2,3-Gal terminated saccharides and SA-α-2,6-Gal terminated saccharides also would be detected by virus histochemistry. We have used this technique previously to show that the pattern of attachment in the human lower respiratory tract is different for human and avian influenza viruses, which correlates with differences in primary disease.¹²     

Production of a Dendritic Cell-Based Vaccine Containing Inactivated Autologous Virus for Therapy of Patients with Chronic Human Immunodeficiency Virus Type 1 Infection

In preparation for a pilot clinical trial in patients with chronic human immunodeficiency virus type 1 (HIV-1) infection, a novel dendritic cell (DC)-based vaccine is being manufactured. The trial will test the hypothesis that isolated endogenous virus presented by DCs serves as a potent immunogen for activation of CD8⁺ and CD4⁺ T cells specific for a broad range of autologous HIV-1 antigens. Production of the vaccine under good manufacture practice conditions involves (i) autologous virus isolation; (ii) superinfection of CD4⁺ T cells with the virus; (iii) inactivation of the virus in CD4⁺ T cells, T-cell apoptosis, and coincubation of T cells with autologous DCs; and (iv) product testing and release. Endogenous virus was isolated from peripheral blood-derived CD4⁺ T cells of three HIV-1-positive subjects by coincubation with autologous OKT-3-stimulated CD4⁺ T cells. CD4⁺ T-cell supernatants were tested for p24 levels by enzyme-linked immunosorbent assay (>25 ng/ml) and for the 50% tissue culture infective doses (TCID₅₀; which ranged from 4,642 to 46,416/ml on day 19 of culture). Autologous CD4⁺ T cells that were separated on immunobeads (>95% purity) and superinfected with virus-expressed p24 (28 to 54%) had TCID₅₀ of >400/ml on days 5 to 10. Virus inactivation with psoralen (20 μg/ml) and UVB irradiation (312 nm) reduced the TCID₅₀ of the supernatants from 199,986 to 11/ml (>99%). 7-Amino-actinomycin D-positive, annexin V-positive CD4⁺ T cells were fed to autologous DCs generated by using the Elutra cell separation system and the Aastrom system. Flow analysis showed that DC loading was complete in 24 h. On the basis of these translational results and experience with the generation of DCs from HIV-1-infected patients in a previous clinical trial, the Investigational New Drug application for clinical vaccination was submitted and approved by the FDA (application no. BB-IND-13137).Antiretroviral therapy (ART) has been widely used to suppress human immunodeficiency virus type 1 (HIV-1) replication and increase the number of CD4⁺ T cells in patients with HIV-1 infection. However, in most of these patients, the recovery of anti-HIV-1-specific T-cell function is incomplete. As the complete restoration of T-cell immune function is considered to be necessary for effective control of the viral infection, additional measures aimed at the bolstering of the HIV-1-specific adaptive immunity in patients treated with ART are being evaluated.Dendritic cells (DCs) are the most potent antigen-presenting cells that can both prime and sustain memory responses (24, 28). DCs have been used increasingly frequently in vaccines against cancer and viral infections (4, 13, 20). Previous studies from our group showed that DCs derived from the blood of subjects with chronic progressive HIV-1 infection and not receiving ART were able to stimulate anti-HIV-1 reactivity (5). HIV-1-reactive CD8⁺ T cells are detectable in the peripheral circulation of subjects receiving ART following in vitro activation with many types of HIV-1 antigens, including HIV-1 proteins, HIV-1 peptides, and virus-infected apoptotic cell-loaded matured DCs (6, 10, 14, 22, 23, 31). We hypothesized that it may be possible to reconstitute the reactivity of naïve and memory virus-specific T cells by delivering to patients autologous DCs engineered ex vivo to express and present known immunodominant peptides of HIV-1. To this end, we have recently completed a phase I clinical protocol in which autologous monocyte-derived DCs were pulsed with a mix of three HIV-1 peptides (Gag, Pol, and Env) and one influenza A virus (matrix) major histocompatibility complex class I supertype peptide and delivered as vaccines to 18 HIV-1-infected, ART-treated subjects (5). This vaccination strategy was found to be safe and feasible and resulted in a transient but significant increase in the frequency of CD8⁺ T cells specific for HIV-1 peptides present in the vaccine (5). On the basis of the results of this trial, we have been considering a strategy of stimulating HIV-1-specific, naïve CD8⁺ and CD4⁺ T cells by priming them with DCs engineered to express autologous HIV-1 (19). The rationale for this strategy is that autologous virus represents a large repertoire of the host''s diverse HIV-1 antigen pool and offers the potential to elicit the most specific, broadest, and most effective immune responses for each subject''s quasispecies of HIV-1, thus increasing vaccine efficacy.In this report, we provide evidence that the production of an antiviral vaccine containing autologous DCs fed with inactivated HIV-1-infected, autologous, apoptotic CD4⁺ T cells is feasible, can be successfully accomplished in a good manufacture practice facility, and can be scaled up for therapeutic delivery to HIV-positive (HIV-1⁺) patients. The production process consists of several steps: (i) isolation of autologous virus from the peripheral blood of HIV-1-infected subjects; (ii) superinfection of autologous enriched CD4⁺ CD8⁻ T cells with viral supernatants; (iii) virus inactivation by psoralen and UVB irradiation; (iv) testing for p24 levels and the residual HIV-1 load by determining the 50% tissue culture infective doses (TCID₅₀) for apoptotic CD4⁺ T cells; and (v) loading of autologous DCs with apoptotic, HIV-1-infected CD4⁺ T cells. Although this process is complex, it has been successfully scaled up for therapeutic vaccine production.     

Changes in the organization of membrane lipids during human platelet activation. Study by fluorescent and freeze-fracture cytochemistry

Modifications in the membrane lipid organization of human platelets activated with different agents (adenosine 5'-diphosphate, thrombin, collagen type I, and monosaccharides such as fucose, mannose, and galactose) were analyzed in vitro by using three lipid markers. Cholesterol was detected upon interaction with filipin, the anionic phospholipids were reacted with polymyxin B, and alterations in the degree of lipid packing were evaluated with the lipophilic fluorescent probe merocyanine 540, which reportedly inserts into bilayer domains whose lipids are more disordered. Filipin-sterol complexes and polymyxin B-anionic phospholipid complexes form characteristic membrane deformations which were examined in freeze-fracture preparations, whereas the merocyanine 540 binding to platelet membrane was recorded by fluorescent microscopy. In contrast to the resting cells, thrombin-stimulated platelets displayed an uneven distribution of filipin-sterol complexes which occurred in much higher density on the cell body than on pseudopods: on the latter, apparently cholesterol-free domains were very common. Unlike the non-stimulated cells, the platelets aggregated with the various agents employed showed characteristic polymixin B-anionic phospholipid complexes deformations of plasmalemma suggesting the appearance in uneven concentration of anionic phospholipids in the outer membrane leaflet. Incubation with merocyanine 540 did not result in staining of resting platelets when these were maintained in plasma, but a slight fluorescence was observed when platelets were kept in Tyrode buffer. However, platelets stimulated with thrombin, collagen type I, and monosaccharides bound very heavily the fluorescent dye; platelets aggregated with adenosine-5'-diphosphate bound only small amounts of merocyanine 540. The results showed that, during activation by different agents, modifications in lipid membrane organization include alterations in cholesterol and anionic phospholipid distribution, transbilayer movement of anionic phospholipids accompanied by more disordered membrane.     

Increased sugar transport in BHK cells infected with semliki forest virus or with herpes simplex virus

Summary Infection of BHK cells by SFV increases the rate of uptake of [³H]MeGlc and of [³H]dGlc at 2 hours p.i. Infection by HSV increases the uptake of [³H]MeGlc and [³H]dGlc at 10 hours p.i.; the increased uptake is prevented by acyclovir. It is concluded that an increased sugar uptake by infected cells reflects an increased rate of transport across the plasma membrane and is the result of cellular changes caused by virus infection.With 5 Figures     

Transfer of the Herpes simplex thymidine kinase gene from human cells to mouse cells by means of metaphase chromosomes

Thymidine kinase (TK)-deficient human cells were infected with ultraviolet light-inactivated Herpes simplex virus type 1, and transformed cells that expressed Herpes TK activity were isolated. Purified metaphase chromosomes were isolated from the transformed human line and incubated with TK-deficient mouse cells. TK ⁺ cells were selected, and it was shown that these cells were gene transferents which expressed Herpes TK activity, identical to that found in the transformed human cells. The gene transferents contained no intact human chromosomes. When removed from selective pressure, the gene transferents rapidly lost the TK ⁺ phenotype. However, upon continued growth in nonselective medium, a subpopulation in which the TK ⁺ phenotype had become more stabilized appeared. These results suggest that the Herpes gene for thymidine kinase has integrated into the genome of the HSV-transformed human cells and that it can be transferred to other cells by means of purified metaphase chromosomes.     

Dengue fever virus and Japanese encephalitis virus synthetic peptides,with motifs to fit HLA class I haplotypes prevalent in human populations in endemic regions,can be used for application to skin Langerhans cells to prime antiviral CD8+ cytotoxic T cells (CTLs)—A novel approach to the protection of humans

Flaviviruses were reported to induce CD8⁺ cytotoxic T cells in infected individuals, indicating that nonapeptides, proteolytic cleavage products of the viral precursor protein, enter the endoplasmic reticulum in infected cells and interact with HLA class I molecules. The assembled HLA class I molecules are transported to the plasma membrane and prime CD8⁺ T cells. Current knowledge of the interaction of viral peptides with HLA molecules is reviewed. Based on this review, an idea is presented to use synthetic flavivirus peptides with an amino acid motif to fit with the HLA class I peptide binding group of HLA haplotypes prevalent in a given population in an endemic area. These synthetic viral peptides may be introduced into the human skin using a lotion containing the peptides (Peplotion) together with substances capable of enhancing the penetration of these peptides into the skin to reach Langerhans cells. The peptide-treated Langerhans cells, professional antigen-presenting cells, may bind the synthetic viral peptides by their HLA class I peptide-binding grooves. Antigens carrying Langerhans cells are able to migrate and induce the cellular immune response in the lymph nodes. This approach to the priming of antiviral CD8⁺ cytotoxic T cells may provide cellular immune protection from flavivirus infection without inducing the humoral immune response, which can lead to the shock syndrome in Dengue fever patients. To be able to develop anti-Dengue virus synthetic peptides for populations with different HLA class I haplotypes, it is necessary to develop computational studies to design HLA class I Dengue virus synthetic peptides with motifs to fit the HLA haplotypes of the population living in an endemic region for Dengue fever. Experiments to study Dengue virus and Japanese encephalitis peptides vaccines and their effectiveness in protection against Dengue fever and Japanese encephalitis are needed. The development of human antiviral vaccines for application of viral peptides in a lotion to human skin (Peplotion) may be useful and affordable for populations of developing countries.     

Nucleic acid synthesis during the focus formation by Shope fibroma virus on green monkey kidney cells

Summary Nucleic acid synthesis during the focus formation on Shope fibroma virus (SFV)-infected cells was studied. When African green monkey kidney (AGMK) cells were infected with SFV, the cell-focus was observed as a local piling up of cells at 3 to 5 days after infection. The number of foci increased in proportion to the size of the SFV inoculum. In the SFV-AGMK cell system, however, the growth rate of virus was comparatively low and no apparent cytopathic effect was demonstrated as a result of virus infection. The incorporation of³H-thymidine into the nuclear fraction of infected cells was suppressed early in the cycle of virus replication and before focus formation. On the other hand, the rate of DNA synthesis increased markedly in the infected cytoplasm. Cytoplasmic RNA synthesis also tended to increase gradually accompanying the induction of the viral DNA synthesis. In addition, the sedimentation properties of the newly synthesized DNA were investigated by sucrose density gradient centrifugation.     

Rearrangement of intermediate filament network of BHK-21 cells infected with vaccinia virus

Summary Association between vaccinia virus (VV) structures and intermediate filaments in specific areas of the cytoplasm of infected cells (virus factories) suggests that VV infection interferes with the cellular architecture by modifying the intermediate filament network. To analyse this question, we examined the array of intermediate filaments of BHK-21 cells infected with VV by laser scanning confocal microscopy using an anti-vimentin mouse monoclonal antibody. We observed a marked reorganization of intermediate filaments around the nucleus of infected cells. Bidimensional analysis of³²PO₄-labeled intermediate filament proteins revealed that the acidic isoform of vimentin and two isoforms of desmin have increased phosphorylation levels in infected cells. Our results suggest that the reorganization of intermediate filaments observed during VV infection could be promoted by an increase in the phosphorylation level of the intermediate filament proteins, vimentin and desmin.