Microarray analysis of amphotericin B-treated THP-1 monocytic cells identifies unique gene expression profiles among lipid and non-lipid drug formulations.

The effects of deoxycholate amphotericin B (DAmB), amphotericin B lipid complex (ABLC), and amphotericin B colloidal dispersion (ABCD) on mRNA profiles from 218 genes in treated THP-1 monocytes were compared. Sixty-one genes were up-regulated and 8 were down-regulated by one or more of the AmB formulations. Fifty-three genes were up-regulated by DAmB while 24 and 18 genes were up-regulated by ABCD and ABLC, respectively. DAmB and ABCD up-regulated many pro-inflammatory genes, whereas ABLC did not. All three formulations up-regulated multiple categories of genes including the anti-apoptosis gene BIRC3 and the chemokine CXCL9 (MIG). Based on representative genes, DAmB activated more signaling pathways than ABCD or ABLC. Quantitative real time PCR confirmed array up-regulation of representative pro-inflammatory genes IL-8, CCL20 (MIP-3alpha), and CXCL2 (MIP-2alpha). Our study represents the first larger scale comparative gene expression profiling which may provide additional rationales for clinical side effects of each amphotericin B formulation.

Selective permissiveness of TPA differentiated THP-1 myelomonocytic cells for human cytomegalovirus strains AD169 and Towne.

Two highly passaged laboratory strains of human cytomegalovirus (HCMV), AD169 and Towne, were tested for their ability to infect and replicate in THP-1 myelomonocytic cells differentiated with 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA treatment of human THP-1 cells increased the number of cells that expressed HCMV immediate early (IE1) antigen from 0.06% prior to TPA treatment to 12% following cell differentiation. The Towne but not the AD169 strain replicated in differentiated THP-1 cells as determined by HCMV DNA replication and infectious virus production. Major early (HCMVTRL4) mRNA was present in both the abortive and productive THP-1 cell infections.

Suppression of LPS-inducible cytotoxicity in cytomegalovirus-infected THP-1 monocytic cell cultures does not correlate with a decrease in TNF-alpha antigen.

We document suppression of tumor necrosis factor alpha (TNF-alpha)-associated cytotoxic activity in a human monocytic cell line (THP-1) infected with the mycoplasma free human cytomegalovirus (CMV) strain AD169. Addition of lipopolysaccharide (LPS) to cell cultures that had been infected with CMV for 24 h resulted in a significant reduction in released cytotoxic activity to mouse L929 cells at 3-22 h post-LPS compared with mock-infected cultures. However, using an ELISA to measure TNF-alpha antigen levels in these culture supernatants, we found infected cultures had significantly higher TNF-alpha antigen levels than in mock-infected cultures following LPS induction. CMV alone also induced TNF-alpha release and possibly TNF-alpha inhibitor(s) which may have blocked TNF-alpha associated cytotoxic activity in CMV-infected THP-1 cell culture supernatants.

Increased monokines in cytomegalovirus infected myelomonocytic cell cultures.

In an investigation of the role of monokines in CMV associated immunosuppression we document modulation of both TNF and IL-1 beta in CMV infected THP-1 cells. CMV infected cultures released almost two-fold more IL-1 beta protein and contained significantly higher IL-1 beta mRNA levels than uninfected cultures for 72-96 h after induction. In both CMV infected and uninfected cultures, significant amounts of IL-1 beta protein were not detected until 24 h post induction, while maximum amounts of TNF were detected in culture supernatants by 3 h post induction, suggesting that TNF may play a role in IL-1 beta induction. TNF levels subsequently declined but in infected cultures remained over 2.5-fold higher than controls through 96 h. The CMV alteration in the kinetics and extent of IL-1 beta release must be indirectly mediated by CMV since only 1% of THP-1 cells were infected. Most infected cells expressed CMV immediate early proteins but did not overexpress IL-1. We speculate that CMV infected cells release excess TNF or other stimulatory factors which increase IL-1 beta synthesis. Since IL-1 beta is increased, the decreased IL-1 'activity' described by others as an explanation in part for the immunosuppressive effects of infection may actually reflect alterations of IL-1 inhibitor levels during CMV infection.

Interactions of human cytomegalovirus with leukocytes in vivo: analysis by in situ hybridization.

Reactivation of human cytomegalovirus (HCMV) from latency occurs in immunosuppressed individuals and infection is itself immunosuppressive. To better understand the basis for this virally induced impairment of immune function, we have analyzed virus-leukocyte interactions by in situ hybridization. We detected viral DNA in 12 viremic patients in the mononuclear cell population, predominantly in cells identified as monocytes by their morphology and by labelling the cells with a monocyte specific monoclonal antibody prior to in situ hybridization. We detected immediate early RNA in infected cells at frequencies comparable to DNA (10(-3) to 10(-5)). By contrast, no viral transcripts were detected in polymorphonuclear cells and viral DNA was inclusively cytoplasmic in accord with the interpretation that this cell type harbors HCMV in phagosomes. These findings in vivo continue to suggest that infection of monocytes plays an important part in the immunosuppressive effects of HCMV infections.

Estimation of the B lymphocyte precursor frequencies to herpes simplex type 1 glycoproteins by a limiting dilution assay.

The precursor frequency of B lymphocytes from Balb/c mice producing HSV-1 glycoprotein B (gB), glycoprotein C (gC), and glycoprotein D (gD) antibody was determined by limiting dilution analysis under conditions to detect antibody from the clonal progeny of a single B cell precursor. In spleens of naive mice the average gC frequency was 1/48,917 +/- 5,550, while gD was 1/73,330 +/- 15,898, and gB frequency was in excess of 1/100,000. Immunization with live HSV-1 (KOS) increased the B cell frequencies of all three glycoproteins to approximately 1:3,000; however, the serum gB antibody ELISA titer was fivefold higher than gC or gD.

A new field strain of equine abortion virus (equine herpesvirus-1) among Kentucky horses.

From restriction endonuclease characterization of the DNA of 317 isolates of equine abortion virus (equine herpesvirus-1; EHV-1) from 176 epizootically unrelated outbreaks of equine virus abortion occurring over 24 years in Kentucky, an epizootic pattern and variation of the virus have emerged. Two electropherotypes of EHV-1 (1P and 1B) accounted for greater than 90% of the nonvaccine-related abortion isolates examined. From 1960 to 1981, EHV-1 1P was the predominant isolate circulating in the central Kentucky area and the cause of greater than 80% of EHV-1-related abortions. In 1981, the occurrence of isolate 1B-related abortions began to increase and since 1982, 1B has become the most frequently recovered isolate of EHV-1 from aborted fetuses.

Identification of the envelope surface glycoproteins of equine herpesvirus type 1.

The structural polypeptides of purified enveloped virions of the Army 183 strain of equine herpesvirus type 1 (EHV-1) were examined by different analytical techniques to identify the envelope glycoproteins. Glycoproteins were identified by electrophoretic analysis in polyacrylamide slab gels of virus labelled in vivo with [3H]glucosamine or labelled enzymically in vitro with either UDP-[14C]galactose or sodium [3H]borohydride. Fluorograms revealed eleven glycoproteins (mol. wt. 260000, 150000, 138000, 90000, 87000, 65000, 62000, 60000, 50000, 46000, and 24000). These glycoproteins probably correspond to virion protein (VP) 1-2, 9b, 10, 13, 14, 16, 17, 18, 21, 22a and 25 respectively, as designated in two other EHV-1 strains. In addition, a poorly resolved glucosamine-rich region (mol. wt. 250000 to 200000) corresponded to VP 3 to 8. The two isotopic surface labelling methods revealed that all the virus glycoproteins were exposed on the envelope surface.

Assessment of the base sequence homology between the two subtypes of equine herpesvirus 1.

The magnitude of the genetic relatedness of the two antigenic subtypes of equine herpesvirus 1 (EHV-1) was determined by DNA-DNA reassociation kinetics. Denatured, labeled viral DNA from one EHV-1 subtype was allowed to reassociate in the presence or absence of the unlabeled heterologous viral DNA. The initial rate of reassociation of either labeled viral DNA was increased by the presence of the heterologous viral DNA to an extent indicating 10 to 20% homology between the two EHV-1 genomes. Similar estimates of the amount of homology between the genomes of the two EHV-1 subtypes were obtained by determining the maximum fraction of labeled viral DNA that could be made resistant to S1 nuclease by hybridization with a large molar excess of the unlabeled, heterologous viral DNA. Analysis of the thermal stability of the subtype 1-subtype 2 heteroduplex DNA indicated approximately 30% base pair mismatching within the hybrid DNA molecules. Cross-hybridization of 32P-labeled virion DNA to nitrocellulose blots of restriction endonuclease cleavage fragments of each EHV-1 subtype DNA indicated that the observed homology between the two viruses was nonuniformly distributed with the viral genome. No homology could be detected between the DNA of either EHV-1 subtype and that of a strain of equine cytomegalovirus (EHV-2). The data suggest that the two biotypes of EHV-1 have arisen by divergent evolution from a common progenitor herpesvirus.

Serologic and molecular comparisons of several equine herpesvirus type 1 strains.

The molecular and serologic relatedness of 2 recent respiratory tract isolates of equine herpesvirus type 1, designated T1 and T2, were compared with the Army 183, Kentucky-A hamster-adapted (KyA-ha), and L-M cell-adapted (KyA-LM) strains. Electrophoresis in polyacrylamide gels revealed differences in virion structural proteins among 4 purified strains. Seven envelope glycoproteins (molecular weight of 93,000, 65,000, 62,000, 60,000, 36,000, 20,000, and 18,000) corresponding to virion proteins 13, 16, 17, 18, 23, 25, and 26a, respectively, found in both the Army 183 and KyA-ha strains had slightly different molecular weight counterparts in both the T1 and T2 isolates, which had identical structural protein profiles. virion protein 19 (58,000 daltons), a nonglycosylated protein, was present in reduced amounts in the respiratory tract isolates, whereas virion protein 8a (200,000 daltons) was absent. Virion protein 8a, an envelope glycoprotein, was only present in the KyA-ha strain. The T1 and T2 isolates were not neutralized by equine herpesvirus type 2 antiserum and revealed little cross-neutralizatio with the Army 183 and KyA-ha strains in plaque-reduction neutralization tests. Restriction endonuclease cleavage maps of viral DNA revealed a similar, but not identical, number and size of DNA fragments between T1 and T2 isolates. Likewise, DNa profiles of Army 183, KyA-ha, and KyA-LM were also similar to each other, but vastly different from the respiratory tract isolates.