Ultra-violet radiation is responsible for the differences in global epidemiology of chickenpox
Background
Of the eight human herpes viruses, varicella-zoster virus, which causes chickenpox and zoster, has a unique epidemiology. Primary infection is much less common in children in the tropics compared with temperate areas. This results in increased adult susceptibility causing outbreaks, for example in health-care workers migrating from tropical to temperate countries. The recent demonstration that there are different genotypes of varicella-zoster virus and their geographic segregation into tropical and temperate areas suggests a distinct, yet previously unconsidered climatic factor may be responsible for both the clinical and molecular epidemiological features of this virus infection.
Presentation of the hypothesis
Unlike other human herpes viruses, varicella-zoster virus does not require intimate contact for infection to occur indicating that transmission may be interrupted by a geographically restricted climatic factor. The factor with the largest difference between tropical and temperate zones is ultra-violet radiation. This could reduce the infectiousness of chickenpox cases by inactivating virus in vesicles, before or after rupture. This would explain decreased transmissibility in the tropics and why the peak chickenpox incidence in temperate zones occurs during winter and spring, when ultra-violet radiation is at its lowest. The evolution of geographically restricted genotypes is also explained by ultra-violet radiation driving natural selection of different virus genotypes with varying degrees of resistance to inactivation, tropical genotypes being the most resistant. Consequently, temperate viruses should be more sensitive to its effects. This is supported by the observation that temperate genotypes are found in the tropics only in specific circumstances, namely where ultra-violet radiation has either been excluded or significantly reduced in intensity.
Testing the Hypothesis
The hypothesis is testable by exposing different virus genotypes to ultra-violet radiation and quantifying virus survival by plaque forming units or quantitative mRNA RT-PCR.
Implications of the hypothesis
The ancestral varicella-zoster virus, most probably a tropical genotype, co-migrated with man as he left Africa approximately 200,000 years ago. For this virus to have lost the selective advantage of resistance to ultra-violet radiation, the hypothesis would predict that the temperate, ultra-violet sensitive virus should have acquired another selective advantage as an evolutionary trade-off. One obvious advantage could be an increased reactivation rate as zoster to set up more rounds of chickenpox transmission. If this were so, the mechanism responsible for resistance to ultra-violet radiation might also be involved in reactivation and latency. This could then provide the first insight into a genetic correlate of the survival strategy of this virus.
Effects of DHEA and Exercise on Strength and Function in Older, Frail Women
Design
Double-blind, randomized, placebo-controlled trial
Participants
Ninety-nine women (mean age 76.6 +/- 6.0) with low sulfated DHEA (DHEAS) levels, low bone mass, and frailty
Intervention
Participants received 50 mg/day DHEA or placebo for 6 months; all received 630 mg/day calcium and 400 IU cholecalciferol. Women participated 90 minutes twice weekly in an exercise regimen.
Primary Outcome Measures
Hormone levels, bone mineral density (BMD), bone turnover markers, body composition, upper- and lower-extremity strength, and physical performance were measured before and after the study.
Key Findings
87 females (88%) completed 6 months. No significant change in BMD or bone turnover marker was noted. Those who received DHEA supplementation with exercise had gains in lower-extremity strength and improvement in Short Physical Performance Battery Score (composite score focusing on lower-extremity function). DHEA was associated with significant changes in all hormone levels, including DHEAS, estradiol, estrone, and testosterone, as well as decreases in sex-binding globulin levels.
Practice Implications
This study reaffirms that DHEA supplementation does not improve BMD nor bone turnover markers. The authors conclude that DHEA supplementation improves lower-extremity strength and function in older, frail women involved in gentle exercise when in fact it is not demonstrated that the supplementation of DHEA had any material impact on performance. Other studies have demonstrated no effect of DHEA on exercise groups.1 While exercise has been well documented to have beneficial improvements in numerous ways, including measurements of biophysical markers and strength, flexibility, and functionality, this study does not provide clear evidence that DHEA supplementation can account for any improvements alone in performance. In this study, it was found that DHEA supplementation combined with exercise in women selected for low DHEAS levels improved lower-extremity muscle strength, which translated to improvement in lower-extremity physical performance.
DHEA supplementation does not consistently improve BMD at all skeletal sites or body composition.
DHEA supplementation does not consistently improve BMD at all skeletal sites or body composition.2,3,4 DHEA supplementation alone for 2 years had no meaningful physiological impact on postmenopausal body composition,5,6 nor did it have any impact on muscle size or strength.7,8 In fact, a study has shown that improvement in strength and physical function only occurred after adding high-resistance training to groups taking supplemental DHEA.9
Further studies need to be completed to evaluate the actual contribution of DHEA supplementation to strength and physical performance over the known benefits of exercise alone.
Histone deacetylase 3. Part 4
We demonstrated that HDAC3 but not HDAC2 interacts with the HCMV MIE locus. The reason that HDAC2 was not detected in our assay could be due to the fact that HDAC2 is known to interact with IE2, and this binding suppresses the repressive effect of HDAC2. At 24 hrs postinfection, the genomic region around the MIE may need to stay repressed, which could be accomplished by the action of HDAC3. This binding of HDAC3 to not only the MIE locus but also the genes upstream of MIE is intriguing. The region between the MIE and UL127 has been named as a unique region without any known function. This region is known to have binding sites for various cellular repressor proteins that help to repress the transcription of UL127. It is known that the promoter of UL127 gene is silenced during productive infection in fibroblasts. The UL126 gene has been reported as a latency-associated gene [29]. UL124 is a putative membrane glycoprotein that may be expressed late in the infection process. It will be interesting to see how the HDACs bind with those genomic regions at later times of infection.
HDAC3 has been shown to be a repressor of the viral MIE promoter. A significant increase in viral replication in the presence of the HDAC inhibitor demonstrates that the binding of HDAC3 causes inhibition of viral replication. This inhibition seems to be more pronounced at a low MOI. Host cells contain several proteins including gene expression suppressors that form a defensive arm against viral infection. HCMV has evolved strategies against cellular defense. We speculate that HCMV infection must reduce HDAC activity. Both IE1 and IE2 of HCMV were reported to functionally interact with HDACs, and IE1 of MCMV is particularly adept at reducing HDAC activity. These observations and our finding of an increase in viral replication in the presence of TSA further confirm the IE2-mediated repression of the MIE promoter by the recruitment of chromatin remodeling factors.
Histone deacetylase 3. Part 2
How HCMV strips off the cellular proteins in order for the virus to replicate its own DNA is not fully understood. Recent studies have shown that HCMV and murine CMV (MCMV) major immediate early proteins, IE1 and IE2 (or IE3 for MCMV), interact with HDAC1, 2, and 3, and HDAC inhibitors enhance viral production, and dynamic chromatin modification of the MIE promoter and other viral promoters has been shown. However, interaction of HDACs with the viral genome has not been clearly demonstrated. In this study, we performed chromatin immunoprecipitation followed by microarray on an HCMV DNA chip (ChIP-on-chip) assay to demonstrate the interaction of HCMV DNA with HDACs. To our surprise, we found that HDAC3, but not HDAC2, interacts specifically with the MIE locus, which suggests a heterogeneous interaction of HDAC3 with HCMV genomic DNA. In addition, we found that the interactions of HDAC3 with the MIE locus might relate to the modulation of viral replication because HDAC3 inhibitors can significantly enhance viral growth.
The chromatinization of viral DNA after its having entered the nucleus has been noted not only in latently infected viruses such as EBV and KSHV (the genomes of which are tethered to cellular chromosomes) but also in the lytic infection of HCMV. On the other hand, histone proteins have not been found in herpesvirus virons. Therefore, the chromatinization of HCMV DNA must be temporary and dynamic. We wonder 1) whether the HDACs are bound to the HCMV DNA, and 2) if so, where they interact and whether the interaction is homogenous or heterogeneous. In order to answer these questions, we performed a ChIP-on-chip assay.
The human foreskin fibroblast cells (HFF) were infected with HCMV at an MOI of 5. The cells were fixed at 24 hours postinfection with 1% paraformaldehyde. The chromatin immunoprecipitation (ChIP) inputs were prepared and performed using the commercial kit (EZ ChIP, Upstate Cell Signal Solutions), according to the manufacturer’s protocol. The antibodies used for ChIP assays include anti-HDAC2 (clone 3F3), anti-HDAC3 (clone 3G6, Upstate USA, Inc.), and normal IgG (as a negative control).
To generate an HCMV genomic microarray for the ChIP-on-chip assay, an entire HCMV (Toledo strain) genomic DNA was subdivided into 593 small DNA fragments and amplified by PCR. Each PCR fragment was ~500 bp long with 100 bp overlapping the adjacent fragments. The PCR products were verified by agarose gels, purified, quantified, and printed on glass slides, as described. Each DNA fragment was spotted in triplicate on each array. The printing quality of the array was controlled by hybridizing the array with a Cyanine 3-dUTP-labeled random 9-mer, and the slides were scanned using an Axon 4000A scanner at 532 nm. Figure 1 shows that all 593 HCMV genomic fragments were printed on the microarray relatively evenly.
Histone deacetylase 3
Evidence suggests that genome chromatinization and the posttranslational modification of histones are involved in the regulation of viral gene expression, including the human cytomegalovirus (HCMV). We performed a ChIP-on-Chip assay to determine whether histone deacetylases (HDACs) interact with HCMV genomic DNA on a global level. Surprisingly, we found that HDAC3, but not HDAC2, interacts not only with the major immediate early (MIE) promoter but also with the entire MIE locus, suggesting a heterogeneous interaction of HDAC3 with HCMV DNA. The interaction of HDAC3 with the MIE region is related to inhibition of viral replication because HDAC3 inhibitors enhanced HCMV replication.
Human cytomegalovirus (HCMV) is a ubiquitous virus and infects a majority of the general population (50-90%). The fact that the incidence of cytomegalic inclusion disease (CID) is intimately related to viral burden suggests that the inhibition of viral production by the specific repression of viral gene expression will reduce the occurrence of CID. Understanding the mechanism of HCMV gene regulation is the pre-requisite for developing drugs that interfere with viral replication by repressing viral gene expression. The HCMV major immediate early (MIE) gene products, IE1 and IE2, are among the first de novo-expressed viral proteins that are required for subsequent viral gene expression and hence viral replication. IE1 and IE2 mRNAs are encoded by the major IE locus that spans from 169 to175 kbp of the viral genome and are produced by alternative splicing and differential polyadenylation. IE1 and IE2 share the first 85 amino acids. MIE genes are controlled by a strong promoter/enhancer that contains many regulatory elements.
Different cellular mechanisms have been found to play roles in inhibiting viral gene expression, and one of the most prominent ones is gene silencing through viral DNA chromatinization (also called chromatin remodeling), a procedure carried out by histone or histone-related proteins, such as histone acetylase (HAT) and histone deacetylase (HDAC). Several posttranslational modifications of histone proteins have been defined to be involved in chromatin remodeling, including acetylation by HAT, deacetylation by HDAC, SUMOylation by SUMO (Small Ubiquitin-like Modifier)-related pathway, deSUMOylation by SENP (a SUMO-specific protease) family enzymes, phosphorylation by kinase pathways, and methylation via methylases. Those enzymatic pathways orchestrate to regulate cellular gene transcription and are termed as epigenetic codes. Viral gene transcription requires cellular machinery, which is probably also regulated by cellular gene regulatory pathways. Histones are abundant nuclear proteins and have been shown to bind with HCMV genomic DNA. Therefore, it was reasonable to propose that chromatin remodeling of viral DNA takes place in the nucleus, which speculation was validated when the fact that HDAC inhibitors can promote cytomegalovirus production was also confirmed.
Herpes simplex virus type 2 infection. Part 4
Twelve hours post-transfection, 293T cells were washed and cultured in RPMI supplemented with 10% FBS. Conditioned medium containing recombinant viruses was harvested and filtered (0.45-μm-pore-size filter) twenty-four hours later. Recombinant viral particles were quantified by reverse transcription (RT) assay. Briefly, virions were precipitated from 1 ml of the filtered supernatants by centrifugation at 13,000 rpm for sixty minutes at 4°C. The precipitate was resuspended in 10 μl of a buffer containing 50 mM Tris-HCl pH 7.5, 1 mM dithiothreitol (DTT), 20% glycerol, 250 mM KCl and 0.25% (v/v) Triton X-100, transferred in dry ice and lysed through three cycles of freezing and thawing. The sample was added to a reaction mixture containing 50 mM Tris-HCl pH 7.5, 7.5 mM MgCl2, 0.05% (v/v) Triton X-100, 5 mM DTT, 100 μg/ml polyA, 10 μg/ml oligo-dT and 2 μCi of 3H-dTTP (43 Ci/mmole) in a final volume of 50 μl. The reaction was incubated for one hour at 37°C and then transferred on Whatman filters. Filters were immediately washed three times in SSC 2× (0.3 M NaCl, 0.03 M sodium citrate pH 7.2) for 10 minutes each, twice in absolute ethanol for ten seconds each and then dried. The radioactivity was measured by using a scintillator (Rackbeta 1214 Wallac) and expressed in counts per million (cpm). In parallel, 1.5 × 106 of purified MDMs were cultured in complete RPMI containing GM-CSF in six-well plates for one week, before being infected with HSV-2 at the MOI of 10 PFU/cell, as previously described. Seventy-two hours later, the cells were transduced with 100,000 H3 cpm RT units of the different HIV-1 recombinant particles previously generated and expressing the CAT reporter gene. Seventy-two hours post-transduction, the MDMs were harvested, lysed in 150 μl of 250 mM Tris-HCl pH 7.5 and then assayed for CAT activity, as previously described. The different forms of acetylated chloramphenicol were separated by thin layer chromatography (TLC) and visualized with an autoradiografic exposure of twelve hours (Kodak Biomax films). The quantitative evaluation was obtained by cutting the TLC paper at the level of the corresponding spots, and by performing a quantification of the spots at the scintillator. The percentage of conversion in the acetylated forms was calculated as follows:
% of conversion = (mono- + di-acetylated forms)/(non acetylated + mono- + di-acetylated forms). Calculated with the above formula, the percentage of conversion is linear for values up to 50%. As expected, the X4-tropic virus (HXBc2 env) does not infect efficiently MDMs, while the dual tropic strain (89.6 env) displays the higher efficiency of infection in all the conditions tested. Our data demonstrate that, following HSV-2 infection, the ability of HIV-1 to superinfect target cells is overall enhanced. The effect is statistically significant (p < 0.05) when R5-tropic recombinant viral particles are examined, correlating this observation, at least partially, with the HSV-2 related increase in CCR5 expression levels on MDMs surface. Not surprisingly, the 89.6 dual tropic strain was extremely efficient in macrophage infection and the observed slight increase in HIV-1 entry upon HSV-2 infection was not statistically significant (p > 0.05).
Herpes simplex virus type 2 infection. Part 3
Thus, R5-tropic strains appear to be the most relevant during HIV-1 acquisition and early phases of infection. Taking into account all these considerations, we focused our attention on HIV-1 CD4 receptor and on the CCR5 coreceptor. 2 × 106 purified MDMs were infected with HSV-2 at the MOI of 10 PFU/cells and, seventy-two hours later, the cells were harvested and analyzed by FACS for the level of CD4 and CCR5 surface expression. In particular, either a CD4 (1:200 v/v, BD Pharmingen) or a CCR5 (1:50 v/v, BD Pharmingen) specific primary antibody, along with a FITC-conjugated anti-rabbit immunoglobulin G secondary antibody (Santa Cruz), were employed on fresh MDMs. Our data show that the percentage of both CD4 and CCR5 positive cells increases after HSV-2 infection (Figure 2B) (p < 0.05). At the same time, also the expression levels of HIV-1 receptor and coreceptor on the cell surface is slightly enhanced, as indicated by the shift in the mean fluorescence intensity displayed by the FACS histograms.
In order to analyze whether the HSV-2 effect on CCR5 expression may have an impact on HIV-1 ability to enter macrophages, we employed a modified version of the previously described env-complementation assay, in which the HIV-1 envelope glycoprotein, expressed in trans, complements a single round of replication of an env-deleted provirus expressing the chloramphenicol acetyltransferase (CAT) gene. Since the defective HIV is capable of only one cycle of replication, this complementation assay allows us to quantitatively measure the abilities of the cells to support the entry of HIV-1 variants containing different envelope glycoproteins, by evaluating the level of CAT expression in the target cells. This assay represents an invaluable tool to dissect the contribution of viral/cellular determinants involved in HIV-1 entry. Recombinant HIV-1 viruses were produced by cotransfection of human embryonic kidney cells (293T, ATCC® Number: CRL-11268TM) with two plasmids, pSVCvpr+vpu+nef+Δenv-CAT and pSVIIIenv. The pSVCvpr+vpu+nef+Δenv-CAT is a derivative of the pSVC21, containing the HIV-1 HXBc2 molecular clone [19], where the vpu, vpr and nef sequences were substituted with those derived from the pNL4-3 (vpu/vpr) and pLAI (nef) molecular clones, in order to introduce functional vpu, vpr and nef genes. Starting from the pSVCvpr+vpu+nef+, we introduced by molecular biology techniques a 580 bp deletion (nucleotides 7041-8621) in the env gene and cloned the chloramphenicol acetyltransferase (CAT), obtained from the v653 RtatC vector, at the BamHI site (nucleotide 8053). The CAT gene is under the transcriptional control of the HIV-1 LTR and is expressed from a subgenomic mRNA generated by the same splicing events used for the natural HIV-1 nef message. Different pSVIIIenv plasmids encoding the HIV-1 Rev protein along with the envelope glycoproteins derived from laboratory-adapted T-cell-tropic (HXBc2), macrophage-tropic (JRF-L and ADA) and primary dualtropic (89.6) HIV-1 isolates, which can use CXCR-4, CCR5 or either one respectively, as a coreceptor, were adopted. Since the viral proteins are expressed in a context similar to that occurring in the authentic provirus, the levels of gene expression achieved are expected to resemble those in HIV-1-infected cells. Briefly, 293T cells were cotransfected by the calcium phosphate method with 20 μg of the pSVCvpr+vpu+nef+Δenv-CAT plasmid and 5 μg of pSVIIIenv plasmids expressing the HIV-1 HXBc2, ADA, JRF-L, or 89.6 envelope glycoproteins to produce recombinant virions. Control viruses lacking envelope glycoproteins were produced by transfecting 293T cells with the pSVCvpr+vpu+nef+Δenv-CAT plasmid alone.
Herpes simplex virus type 2 infection. Part 2
Thus, in order to analyze whether the low susceptibility to HSV-2 infection displayed by U937 cells could be related to their differentiation level, the cells were induced to differentiate by treatment with TPA (50 ng/ml). After twelve hours of incubation, U937 cells were washed twice with PBS and cultured for additional twenty-four hours in TPA-free medium, in order to avoid possible effects of residual TPA. The percentage of cells positive for CD14 surface expression, a marker of macrophage differentiation, was then determined by Fluorescence-Activated Cell Sorting (FACS). Briefly, 1 × 106 cells were harvested and directly incubated for one hour in cold PBS containing 1:100 (v/v) of an anti-human CD14 primary antibody (Li StarFISH). A fluorescein isothiocyanate (FITC)-conjugated anti-rabbit immunoglobulin G antibody (Santa Cruz) was employed as secondary antibody and the fluorescence was evaluated by FACS analysis (FACScalibur, Beckton Dickinson). As reported in Figure 1B, after TPA treatment the percentage of CD14-positive U937 cells is significantly increased. Differentiated U937 cells were infected with HSV-2 (MOI of 1 PFU/cell). Infectious virus yields, which peaked approximately three days post-infection, appear to be significantly higher than those obtained from undifferentiated U937 cells. Thus, our data suggest that HSV-2 replication efficiency is dependent on the differentiated phenotype of U937 cells along the monocytic pathway. Interestingly, while untreated U937 cells did not display a significant HSV-2 induced cytopathic effect (CPE), TPA-differentiated U937 cells were fully susceptible to viral CPE (data not shown).
Next, infection of primary monocyte-derived macrophages (MDMs) was performed. MDMs were obtained from at least three pooled buffy coats of HIV-1 seronegative healthy blood donors, by Ficoll-Hystopaque gradient, followed by plastic adherence of PBMCs for sixteen hours in complete RPMI. Non-adherent cells were removed, and adherent cells were extensively washed with PBS and grown in complete RPMI supplemented with Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF, 500 U/ml). The cells were cultured for one week, before evaluating the preparation purity by measuring the percentage of CD14-positive cells through FACS analysis, as described above. The cut-off employed to accept the purity of MDM preparation was a CD14-positive percentage higher than 90%. Purified MDMs (2 × 106) were infected with HSV-2 strain G at two different MOIs, 10 PFU/cell and 1 PFU/cell, following the same experimental procedure described for the U937 cell line. After viral adsorption, the infected MDMs were maintained in complete RMPI medium supplemented with GM-CSF. Our data show that MDMs support HSV-2 replication. As expected, at the MOI of 10 PFU/cell the infection appears to be more efficient, achieving a peak in the viral titre seventy-two hours post-infection. No clear viral induced CPE could be observed at any time post-infection (data not shown). Next, we analyzed the effect of HSV-2 infection on HIV-1 receptor and coreceptors expression on macrophage cell surface. Indeed, HIV-1 entry into target cells involves the interaction of the viral envelope glycoproteins with the host cell receptors, CD4, and one of two coreceptors CCR5 or CXCR4. The chemokine receptors CXCR4 and CCR5 represent indeed the coreceptors for T-cell-tropic (X4-tropic) and macrophage-tropic HIV-1 (R5-tropic) strains, respectively. Dual/mixed-tropic populations are also present in HIV infected patients and evidence has been collected in antiretroviral-naive patients suggesting that the majority of viruses within these dual/mixed-tropic populations use CCR5. Moreover, natural history studies of HIV-1 infection have shown that most patients harbour R5-tropic virus populations soon after infection and through the asymptomatic phase.
Herpes simplex virus type 2 infection
Abstract
Epidemiological and clinical data indicate that genital ulcer disease (GUD) pathogens are associated with an increased risk of human immunodeficiency virus type 1 (HIV-1) acquisition and/or transmission. Among them, genital herpes simplex virus type 2 (HSV-2) seems to play a relevant role. Indeed, the ability of HSV-2 to induce massive infiltration at the genital level of cells which are potential targets for HIV-1 infection may represent one of the mechanisms involved in this process. Here we show that infection of human primary macrophages (MDMs) by HSV-2 results in an increase of CCR5 expression levels on cell surface and allows higher efficiency of MDMs to support entry of R5 HIV-1 strains. This finding could strengthen, at the molecular level, the evidence linking HSV-2 infection to an increased susceptibility to HIV-1 acquisition.
Findings
Herpes simplex virus (HSV), and especially HSV type 2, represents one of the most widely spread pathogen causing genital ulcer disease (GUD). Different studies have associated GUD aetiological agents in general, and HSV-2 in particular, with a higher risk to acquire and/or transmit HIV-1 infection. A number of biological and molecular factors may explain this evidence. Both the physical disruption of the epithelial/mucosal barrier and the cellular inflammatory response characterizing GUD could facilitate HIV-1 acquisition, by providing the virus with access to a large number of CD4-positive cells. Moreover, several in vitro studies have underlined molecular mechanisms by which HSV can directly influence the HIV life cycle in HSV-HIV coinfected cells. Finally, randomised controlled trials have been conducted in coinfected individuals to evaluate the effect of HSV-2 suppressive therapy on HIV-1 genital shedding and plasma HIV-1 RNA, showing, in most cases, a negative impact on HIV-1 replication. A recent study conducted by Zhu and co-workers showed a persistence of HIV receptor-positive cells in genital skin after HSV reactivation. In the genital tract, macrophages represent one of the main target of HIV-1, especially during primary infection. In this study we wanted to analyze the ability of HSV-2 to infect human macrophages and to influence HIV-1 super-infection.
Firstly, we selected the human monocyte U937 cell line (ATCC® Number CRL-1593.2) as experimental set and we infected them with HSV-2, strain G (kindly provided by Dr. Peggy Marconi, University of Ferrara, Italy). Briefly, the virus was grown and titrated by plaque assay on African green monkey kidney cells (Vero), as previously described. U937 cells (1 × 106) were infected with HSV-2 at two different multiplicity of infection (MOI of 1 and 10 plaque forming unit, PFU, per cell). Cells were left in contact with the virus for two hours at 37°C and, after three washing with phosphate buffered saline (PBS), cultured in Roswell Park Memorial Institute medium (RPMI 1640), with addition of 10% heat-inactivated foetal bovine serum (complete medium). HSV-2 replication was followed by titration of the virus released in the cellular supernatant. In contrast with fully permissive Vero cells, our data show that U937 cells do not support a significant HSV-2 replication and that, at least in the case of the MOI of 1 PFU/cell, the viral titre declines over time. It has been previously reported that monocytes display an intrinsic resistance to HSV type 1 (HSV-1) infection, depending on the cellular differentiation level along the monocytic pathway into functionally and morphologically mature non-proliferating cells, that can be achieved by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment.
Delayed-type hypersensitivity (DTH) reaction to rotavirus EDIM and RRV. Conclusion
Much controversy still exists as to whether serum antibodies against rotavirus are directly involved in protection or merely reflect recent infection, leaving the protective role to mucosal or cell-mediated immunity. Reviewed data from a variety of studies in humans suggest that serum antibodies, if present at critical levels, are either protective themselves or are an important and powerful correlate of protection against rotavirus disease. Previous studies in infant mice, rabbits and humans have determined that rotavirus-specific IgM levels increase during the acute-phase of infection (before 7 days p.i.) and then decrease gradually. Therefore, rotavirus-specific IgM is seen as a marker of primary infection. Rotavirus-specific IgA and IgG levels were increased in the convalescent-phase of the infection. Administration of RRV at day 7 resulted in the development of an antibody titer after 21 days, predominantly of the IgG2a subclass. Similar subclass restriction after virus infections was seen previously. Administration of EDIM to previously RRV inoculated mice did not result in an increase of the antibody titer. Administration of EDIM at day 17 without a previous RRV inoculation resulted in normal levels of rotavirus-specific IgM and a small amount of rotavirus-specific IgG (subclass) antibodies, most likely due to the fact that 10 days p.i. is too early to measure the development of IgG antibodies. Intervention with Gastrogard-R® showed low antibody titers, but still significantly higher than the group who were inoculated with EDIM only, indicating that the rotavirus, although not able to induce diarrhea during the primary infection, could still provoke an antibody reaction.
Gastrogard-R® is prepared from colostrum of hyperimmunised cows and contains high antibody titers against four human rotavirus serotypes, as measured in a virus neutralisation test. The efficacy of passive immunization was established in calves which were immunized by subcutaneous injection of colostral whey with a high IgGl rotavirus antibody titer and challenged with virulent bovine rotavirus 48 h later. Calves were protected from rotavirus infection and diarrhea. Results further indicated that circulating IgG1 antibody appeared in the gastrointestinal tract of neonatal calves. Previous research already showed that rotavirus antibody activity survived passage through the human gastrointestinal tract [28]. Gastrogard-R® is used as prophylactic treatment of ‘at risk’ children aged one month to three years to prevent diarrhea due to rotavirus infection. The efficacy of treatment with Gastrogard-R® was established in a clinical trial in children aged 3 to 15 months. The in vitro inhibitory effect of Gastrogard-R® was established in our laboratory in a rotavirus (RRV) titration assay using MA-104 cells (data not shown). In this assay Gastrogard-R® was shown to have a strong inhibitory effect on the infectivity of rotavirus with an IC50 of 1 μg/ml. Bovine milk and bovine milk constituents like lactadherin have been studied on their inhibitory activity in vitro and in in vivo rotavirus models. Various compounds present in whey protein concentrate can ameliorate the severity and incidence of experimental rotaviral diarrhea and modulate the mucosal and systemic immune in suckling rats and suckling mice. However, none of these dairy compounds were able to completely inhibit clinical symptoms during a primary rotavirus infection, which is usually attended with the most severe clinical symptoms like diarrhea and vomiting.
Conclusions
In this study was found that oral administration of rotavirus antibodies can completely protect neonatal mice from clinical symptoms of illness during a primary rotavirus infection. Furthermore, an enhanced rotavirus-specific T cell proliferation and a small but detectable level of rotavirus-specific antibodies were found after re-infection suggesting improved T cell responses and a slight B cell response. Also shedding of rotavirus after EDIM inoculation seemed to disappear more rapidly in mice treated with rotavirus antibodies than in mice inoculated with EDIM alone. These results indicate that even though symptoms of a primary rotavirus infection were prevented, an activation of the immune system was still detectable. Preventing a primary infection by using Gastrogard-R®, activation of the immune system can still occur and could be helpful during re-infection knowing that both arms of the immune system play a pivotal role in immunity to rotavirus infection. These data show that this intervention model can be used for studying clinical symptoms as well as immune responses required for protection against viral re-infection.