Cytomegalovirus infection causes morbidity and mortality in patients with autoimmune diseases, particularly systemic lupus: in a Chinese population in Taiwan.

To investigate the clinical outcome of cytomegalovirus (CMV) infection in febrile hospitalized patients with autoimmune diseases, mostly systemic lupus erythematosus (SLE). Fifty-four febrile patients were analyzed retrospectively. Half were diagnosed as CMV infection, by positive CMV pp65 antigenemia assay. Clinical and laboratory data between two groups were compared. Correlation between laboratory data and SELENA-SLEDAI scores/mortality were analyzed in the CMV infection group. Receiver operating characteristic analysis was performed to determine the cutoff points of different parameters for predicting mortality or morbidity. The CMV infection group received a higher corticosteroid dosage (mean 26.3 mg/day) and a higher percentage of azathioprine use before admission than the non-CMV infection group. In the former, the deceased subgroup had a significantly higher number of infected leukocytes for CMV (shortened as CMV counts, P = 0.013), more cases of bacterial infection (P = 0.090), and a higher SLE disease activity index score (P = 0.072) than the alive subgroup. The CMV infection group had lower lymphocyte count and more positive bacterial infection than the non-CMV infection group did (P = 0.013 and P = 0.027, respectively). A level of 25 CMV particles/5 × 10(5) polymorphonuclear neutrophils (PMN) was the best cutoff point for predicting CMV-associated mortality, with a sensitivity of 75.0% and specificity of 72.2%. Moderate dose (30 mg/day) of prednisolone or azathioprine use predisposes patients with autoimmune diseases to CMV infection with concurrent bacterial infection. In particular, peak CMV counts at 25/5 × 10(5) PMN or low lymphocyte counts predict mortality or morbidity, respectively.

Viremia control following antiretroviral treatment and therapeutic immunization during primary SIV251 infection of macaques.

Prolonged antiretroviral therapy (ART) is not likely to eradicate human immunodeficiency virus type I (HIV-I) infection. Here we explore the effect of therapeutic immunization in the context of ART during primary infection using the simian immunodeficiency virus (SIV251) macaque model. Vaccination of rhesus macaques with the highly attenuated poxvirus-based NYVAC-SIV vaccine expressing structural genes elicited vigorous virus-specific CD4 + and CD8+ T cell responses in macaques that responded effectively to ART. Following discontinuation of a six-month ART regimen, viral rebound occurred in most animals, but was transient in six of eight vaccinated animals. Viral rebound was also transient in four of seven mock-vaccinated control animals. These data establish the importance of antiretroviral treatment during primary infection and demonstrate that virus-specific immune responses in the infected host can be expanded by therapeutic immunization.

Inhibition of human immunodeficiency virus infectivity by chloroquine.

The effect of chloroquine, a drug known to affect intracellular exocytic pathways, was studied in two retroviral systems: human immunodeficiency virus (HIV-1) and avian reticuloendotheliosis virus (REV-A). With chloroquine treatment of virus-infected cells, significant size reduction of the cell- and virus-associated surface glycoproteins, gp90 of REV-A and gp120 of HIV-1, was observed. In the case of HIV-1, extracellular virions derived from treated cells contained very little gp120. Infectivity and reverse transcriptase assays of HIV-1 demonstrated that by chloroquine treatment the majority of the virions released was noninfectious and the total virus yield was also reduced. The data suggest that chloroquine inhibition of infectious virus production is most likely due to interference with terminal glycosylation in the trans-Golgi network.

The presence and absence of histocompatibility antigens in HIV type 1 produced by autologous blood-derived macrophages and peripheral blood lymphoblasts.

Acquisition of cellular proteins by HIV-1 virions is known to alter the physiology of the virus in vitro. Reported studies of this aspect have been largely limited to transformed T cell lines. In this study, we investigated the incorporation of major histocompatibility antigens (HLAs) on a primary macrophage-tropic isolate, HIV-1ADA, grown from autologous monocyte-derived macrophages (MDMs) and peripheral blood mononuclear cells (PBMCs). A virus precipitation assay (VPA) demonstrated that HIV-1ADA grown from PBMCs incorporated substantial amounts of HLA class I (alpha chain and beta2m) and DR antigens, comparable with a laboratory strain, HIV-1MN, grown from the same host cells. HIV-1ADA, however, grown from MDMs incorporated significantly lower amounts of HLAI and -II antigens despite the fact that the infected MDMs were found to express significant amounts of HLA antigens. The lack of incorporation of these important immunomodulatory cell surface proteins may be yet another unique characteristic of macrophage-tropic isolates and suggests a possible role in their biology and or immunology.

Preliminary findings of an in vitro human spleen mononuclear cell culture system for primary isolates of HIV type 1.

Acute HIV-1 infection is often manifested with a high level of viremia. The cell types and tissues/organs that contribute to the virus load are thought to be of central and peripheral lymphoreticular origin. The establishment and permissiveness of organ-based cell culture systems from spleen with laboratory strains or primary isolates of HIV-1 have not been reported. We studied unseparated splenic mononuclear cells (SMCs) and adherent cells derived from human spleen and liver in comparison with blood monocyte-derived macrophages (MDMs). Unstimulated, SMCs were highly permissive to primary lymphotropic HIV-1 and dual/macrophage-tropic isolates (which are able to replicate in both MDMs and PBMCs). Furthermore, SMCs were found to replicate virus to high titer in a rapid log-phase manner and exhibited a prolonged stationary phase of virus production, unlike PBMCs, which required conventional activation with mitogens and exhibited a much shorter period of virus production. Interestingly, the SMCs maintained themselves as a mixed phenotype of nested lymphocytes with complex and well-differentiated macrophage(s) for extended periods of time. In addition, splenic macrophages readily purified by adherence were highly permissive to a dual/macrophage-tropic primary isolate, HIV-1ADA, intermediate with two laboratory strains, HIVR-1RF and HIV-lHXB3, and least permissive to the lymphotropic primary isolate HIV-1Mr452 and two other laboratory strains, HIV-1CC and HIV-1MN. The replication of HIV-1ADA as measured by extracellular p24 was sustained for up to 7 weeks and similar to the replication patterns observed with adherent hepatic macrophages and blood-derived MDMs. This study demonstrates that exogenous stimulation is not required for infection of these cells; either adherence-isolated and/or mixed lymphoid populations can be studied together, and viable stocks can be readily prepared and cryopreserved. In addition, these cells could be used for isolating new and/or other variants of HIV-1. Thus, the use of the SMC primary in vitro cell culture system for future studies involving HIV-1 is warranted.

Design and in vivo immunogenicity of a polyvalent vaccine based on SIVmac regulatory genes.

Most vaccine modalities for human immunodeficiency virus type 1 (HIV-1) tested for immunogenicity and efficacy in the SIVmac (simian immunodeficiency virus) macaque model do not include the viral regulatory proteins. Because viral regulatory proteins are expressed early during the virus life cycle and represent an additional source of antigens, their inclusion as a vaccine component may increase the overall virus-specific immune response in vaccinees. However, at least two of the early proteins, Tat and Nef, may be immunosuppressive, limiting their usefulness as components of an SIV vaccine. We have constructed a polyvalent chimeric protein in which the open reading frames for Tat and Nef have been reassorted and the nuclear localization sequence for Tat and Rev and the myristoylation site for Nef have been removed. The resulting DNA plasmid (pDNA-SIV-Retanef) (pDNA-SIV-RTN) encodes a protein of 55 kDa (Retanef) that localizes at the steady state in the cytoplasma of transfected cells. Both the DNA-SIV-RTN and the highly attenuated recombinant poxvirus vector NYVAC-SIV-RTN were demonstrated to be immunogenic in SIVmac251-infected macaques treated with ART as well as in naive macaques. An equivalent strategy may be used for the generation of polyvalent antigens encoding the regulatory proteins in a HIV-1 vaccine candidate.

Matrix metalloproteinase-8 in sera and from polymorphonuclear leucocytes in rheumatoid arthritis: in vitro characterization and correlation with disease activity.

OBJECTIVES: To determine matrix metalloproteinase-8 (MMP-8) secretion from rheumatoid arthritis (RA) peripheral blood polymorphonuclear leucocytes (PMNs), in response to immune complexes (IC), cytokines and their combinations, and to study correlation of serum MMP-8 with disease activity. METHODS: PMNs from RA patients and controls were stimulated in vitro with interleukin-15 (IL-15), IL-18, adherent immune complexes, rabbit anti-human immunoglobulin G (anti-HIgG), human immunoglobulin G (HIgG), and their F (ab') 2 prongs, phorbol myristate acetate (PMA) or combinations of above. Supernatants from these experiments and sera from both groups were assayed for MMP-8 using ELISA and correlated with disease activity measures in patients. RESULTS: MMP-8 secretion from stimulated PMNs was compared to unstimulated PMNs. Immune complexes elicited significant MMP-8 secretion (p = 0.006 and 0.001, control and RA respectively). Unlike HIgG and its F (ab')2 fragment, very high secretion was elicited by anti-HIgG (242.37 +/- 10.85 ng/ml) and its F (ab')2 prong (195.85 +/- 28.67 ng/ml). IL-15 did not elicit any secretion. IL-18 with PMA increased secretion significantly only from RA PMNs (p = 0.003). Serum MMP-8 correlated positively with serum CRP (p = 0.017) and not with disease activity score (p = 0.199). CONCLUSIONS: We for the first time demonstrate that immune complexes elicit MMP-8 secretion from PMNs. Except for higher secretion from RA PMNs in response to combination of IL-18 and PMA, both control and RA PMNs respond similarly to various stimuli. Secretion by anti-HIgG occurs by a mechanism independent of Fc receptor. Correlation with CRP suggest that serum MMP-8 may be an indicator of acute inflammatory activity.

[Hypothyroid myopathy: a case report].

Though proximal muscle weakness is characteristic of polymyositis, other agents may also lead to proximal muscle weakness, such as drugs, endocrine diseases, or infections. Here, we report a thirty year-old female suffering from proximal weakness which initially was thought to be a case of polymyositis with high serum creatine phosphokinase level. Very low thyroid hormone levels were found as the history inquiry revealed constipation, hoarseness and cold intolerance. The muscle biopsy showed no obvious inflammation. After thyroxine therapy, her muscle weakness recovered. Up to now, hypothyroid myopathy with muscle mitochondrial abnormalities shown by electron microscopic examination has not been reported in the medical literature in Taiwan. Hypothyroid myopathy, though it is rare, may be misdiagnosed as polymyositis clinically. Therefore, it is recommended that hypothyroid myopathy should be considered in the differential diagnosis of proximal muscle weakness.

Altered balance between Th17 and Th1 cells at mucosal sites predicts AIDS progression in simian immunodeficiency virus-infected macaques.

Loss of CD4(+) T cells in the gut is necessary but not sufficient to cause AIDS in animal models, raising the possibility that a differential loss of CD4(+) T-cell subtypes may be important. We found that CD4(+) T cells that produce interleukin (IL)-17, a recently identified lineage of effector CD4(+) T-helper cells, are infected by SIV(mac251)in vitro and in vivo, and are found at lower frequency at mucosal and systemic sites within a few weeks from infection. In highly viremic animals, Th1 cells predominates over Th17 T cells and the frequency of Th17 cells at mucosal sites is negatively correlated with plasma virus level. Because Th17 cells play a central role in innate and adaptive immune response to extracellular bacteria, our finding may explain the chronic enteropathy in human immunodeficiency virus (HIV) infection. Thus, therapeutic approaches that reconstitute an adequate balance between Th1 and Th17 may be beneficial in the treatment of HIV infection.

CD4+ T-cell loss and delayed expression of modulators of immune responses at mucosal sites of vaccinated macaques following SIV(mac251) infection.

Systemic immunization of macaques with a combination of DNA-poxvirus-based vaccines confers protection from high level of both systemic and mucosal viral replication following rectal exposure to the pathogenic SIV(mac251). Here we investigated early post-infection events in rectal and vaginal tissues, and found that the loss of CCR5+CD4+ T cells was equivalent in vaccinated and control macaques, despite a three logs reduction at mucosal sites of simian immunodeficiency virus (SIV) RNA in the vaccinated group. Even though a normal CD4+ T cell number is not reconstituted at mucosal sites in either group, vaccination appeared to confer a better preservation of the CD4+ CCR5+ T cells that replenish these sites. Analysis of rectal tissues RNA following challenge exposure demonstrated a decreased expression in vaccinated macaques of transforming growth factor-beta, cytotoxic T lymphocyte antigen-4, FoxP3, and indoleamine 2,3-dioxygenase, an immune suppressive enzyme expressed by dendritic cells that converts tryptophan to kynurenine and limits T-cell responses. Accordingly, the ratio of kynurenine and tryptophan in the plasma was significantly reduced in the vaccinated animals respect to the controls. Thus, preexisting adaptive immune responses induced by these vaccine modalities, although they do not protect from CD4+ T-cell depletion, nevertheless, they contain SIV(mac251) replication and delay expression of markers of T-cell activation and/or suppression at mucosal sites.

Invasive fungal infection in systemic lupus erythematosus: an analysis of 15 cases and a literature review.

OBJECTIVE: To analyse 15 cases of invasive fungal infection and mortality parameters in the largest series in the last 35 yrs of patients with systemic lupus erythematosus (SLE) at a single medical centre. METHODS: Fifteen patients with SLE and invasive fungal infections were retrospectively enrolled. Clinical and laboratory data, fungal species and infected sites, corticosteroid and immunosuppressant doses and SLE disease activity index were assessed retrospectively. Comparison and correlation analyses utilized Fisher's exact test, the chi-square test, Mann-Whitney U-test or the Wilcoxon signed-rank test where appropriate. RESULTS: In contrast to other review reports, Cryptococcus neoformans was the most commonly identified fungus in this Taiwanese series. Notably, the prevalence of autoimmune haemolytic anaemia and positive results for the anti-cardiolipin antibody in this study were significantly higher than those in SLE patients in general (P < 0.0001 and P < 0.0001, respectively). Fungal infection contributed to cause of death in 7 of 15 (46.7%) patients, of which Cryptococcus neoformans accounted for six of these infections. Low-dose prednisolone (<1 or <0.5 mg/kg/day based on arbitrary division) prior to fungal infection tended to correlate with 1 yr mortality after diagnosis of SLE (P = 0.077 or P = 0.080). However, following fungal infection, patients who died from infection itself had been prescribed with higher prednisolone dose or equivalent than surviving patients (P = 0.016). All SLE patients with fungal infections had active SLE (SLEDAI >7). CONCLUSIONS: Cryptococcus neoformans infection accounted for most fatalities in SLE patients with fungal infections in this series. Active lupus disease is probably a risk factor for fungal infection in SLE patients. Notably, low prednisolone doses prior to fungal infection or high prednisolone doses following fungal infection tended to associate with or correlated to fatality, respectively. Therefore, we suggest that different prednisolone doses prescribed at various times impact the incidence of fungal infection and its associated mortality.

Symptomatic renal tubular acidosis (RTA) in patients with systemic lupus erythematosus: an analysis of six cases with new association of type 4 RTA.

OBJECTIVES: We have analysed the association between different parameters of renal tubular acidosis (RTA) with clinical and laboratory parameters in patients with systemic lupus erythematosus (SLE). METHODS: Review of hospital database records between 1978 and 2003 revealed six SLE patients with RTA. Correlations and comparisons were done by Spearman rank correlation coefficient and the chi(2) test. RESULTS: Four patients had hypokalaemia (type 1 RTA) and two patients had hyperkalaemia (type 4 RTA). Three patients with type 1, but no patients with type 4 RTA, had medullary nephrocalcinosis. The majority of SLE patients with distal RTA (type 1 and type 4) had nephritis with proteinuria. No seronegative SLE was noted, and all patients were negative for anticardiolipin antibodies. There was a noticeable trend of higher serum potassium levels with increased SLE Disease Activity Index (SLEDAI; P < 0.1) and nephritic manifestation (haematuria, P < 0.1). The mean SLEDAI scores were 11.75 and 27.5 for type 1 and type 4 RTA patients, respectively. CONCLUSIONS: When present in patients with SLE, classic distal RTA (type 1) is the most common. In particular, we report here for the first time two cases of type 4 RTA in SLE patients with higher SLEDAI scores than patients with type 1 RTA. Medullary nephrocalcinosis or renal urolithiasis has not been found in our patients with type 4 RTA. Higher serum potassium levels seem to be associated with higher SLEDAI scores and more severe nephritic manifestations in patients with distal RTA.

Site-directed cytotoxic antibody against the C-terminal segment of the surface glycoprotein gp90 of avian reticuloendotheliosis virus.

The major mature env-gene products of avian reticuloendotheliosis-associated virus (REV-A) are the surface glycoprotein (gp90) and the transmembrane protein (gp20). We have previously reported that gp90 was detected in the REV-A virus by Western blot analysis as well as in the REV-A-infected cells by radioimmunoprecipitation with antibodies raised in rabbits against the gp90 C-terminal tridecapeptide which was predicted from the nucleotide sequence (Wilhemsen et al., J. Virol., 52, 172, 1984). We have now shown that this antibody detected antigens on the REV-A-infected cells by fluorescence-activated cell sorter (FACS) analysis, and conferred specific cytotoxic effects on the infected cells in the presence of rabbit complement using the chromium release assay. These results clearly indicate that the C-terminal epitope of gp90 is situated on the surface of the REV-A-infected cells and accessible to site-directed antibodies which cause cytotoxicity by activating the complement system. The possible in vivo roles of this antibody are discussed.

Novel glycosylation pathways of retroviral envelope proteins identified with avian reticuloendotheliosis virus.

Previously, we identified two mature glycoproteins, gp90, the surface glycoprotein, and gp20, the transmembrane protein, from avian reticuloendotheliosis virus and an avian reticuloendotheliosis virus env gene-encoded intracellular polyprotein gPr77env, but the precise relationship of gPr77env to the mature envelope proteins was not determined (W.-P. Tsai, T.D. Copeland, and S. Oroszlan, Virology 155:567-583, 1986). In the present study, using metabolic labeling of viral proteins with [35S]cysteine, radioimmunoprecipitation, and carbohydrate structure analysis, we have identified a higher-molecular-weight endo-H-resistant env gene-encoded polyprotein designated gPr115env in addition to the endo-H-sensitive gPr77env. It appears that gPr77env is the primary polyprotein precursor, modified with mannosyloligosaccharides that are processed into sialic-acid-rich extraordinarily large complex-type carbohydrates (up to 17 kilodaltons for each N-linked site) on the gp90 domain but not on the gPr22 domain. In this process, gPr77env is converted into the apparently endo-H-resistant secondary polyprotein, gPr115env, which is rapidly processed into gp90 and gPr22. The proteolytic processing which occurs only after the appearance of an endo-H resistant precursor is now clearly demonstrated for a retrovirus. Some important aspects of carbohydrate structure, including the site-specific glycosylation, as well as the intracellular location and nature of the potential enzyme involved in the proteolytic cleavage of gPr115env are discussed.

Purification and chemical and immunological characterization of avian reticuloendotheliosis virus gag-gene-encoded structural proteins.

Five gag-gene-encoded structural proteins, designated p12, pp18, pp20, p30, and p10 were purified from replication-competent avian reticuloendotheliosis-associated virus (REV-A) by high-performance liquid chromatography complemented with chloroform-methanol extraction and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based on amino acid composition and NH2- and COOH-terminal sequence analysis p12, pp18, p30, and p10 are distinct from one another, whereas pp20 is likely identical to pp18 in primary structure. The p12 was resistant to Edman degradation and was found to be myristylated at the NH2-terminal amino group. Sequence comparisons among the retrovirus family show that pp18/pp20 and p10 are, respectively, homologs of phospho-proteins and nucleic acid-binding proteins. A comparison of terminal sequences with the nucleotide sequence of spleen necrosis virus (SNV) revealed that the gag genes of SNV and REV-A are highly conserved; together with the identification of REV-A gag-precursor polyprotein, Pr60gag in immunoprecipitates of radiolabeled cell lysates, this comparison also led to the establishment of the organization of Pr60gag, viz., NH2-p12-pp18-p30-p10-OH. Sequence comparisons show that REV-A/SNV is related to mammalian type C viruses: the pp18-p30 region is most homologous to the macaque/colobus group and least to simian sarcoma virus (SSV), whereas both the 5'- and 3'-gag regions (i.e., p12 and p10) are clostest to SSV. Immunological studies using monospecific antisera and Western-blot analysis showed that antigenic determinants of REV-A p30 are conserved in most of mammalian type C and type D viruses, but those of REV-A p12 are shared only with simian sarcoma-associated virus (SSAV) and endogenous viruses of macaques.

Biosynthesis and chemical and immunological characterization of avian reticuloendotheliosis virus env gene-encoded proteins.

Two glycosylated proteins designated gp90 and gp20 were purified from replication-competent avian reticuloendotheliosis associated virus (REV-A). The N-terminal sequences of gp90 and gp20 were determined and found to match the REV-A-env-gene sequence. The alignments of the determined amino acid sequences with the predicted sequence indicate that gp20 and gp90 are the REV-A-encoded viral transmembrane and surface glycoprotein, respectively, and predict a signal peptide of 36 residues on the 5' end of the env-gene. Furthermore, gp90 of REV-A was detected by Western blot analysis with antibodies to a tridecapeptide corresponding to an env-gene nucleotide segment immediately preceding gp20 and thus representing the C-terminal portion of gp90. The env-gene precursor polyprotein gPr75-79env and Pr22(E), the precursor to gp20 and p2(E) were identified in the infected cells by monospecific antibodies raised against purified gp20. Thus the organization of gPR75-79env is likely to be N-gp90-gp20-p2(E), resembling that of M-MuLV gp85env. Sequence comparisons showed that the env gene of REV-A is highly related to both baboon endogenous virus and Type D retroviruses. In Western blot analyses, antibodies to REV-A gp20 cross-reacted with a panel of mammalian Type C and Type D viruses. Evolutionary aspects of these findings are discussed.

Antibody to a synthetic peptide detects a conserved region of retrovirus transmembrane proteins.

N-terminal amino acid sequence analysis of the transmembrane protein of baboon endogenous virus revealed an internal 13 residue identity with the transmembrane homolog of murine leukemia virus. A tridecapeptide Glu-Val-Val-Leu-Gln-Asn-Arg-Arg-Gly-Leu-Asp-Leu-Leu corresponding to this region was chemically synthesized and antibody to the peptide was raised in rabbits. The rabbit antisera recognized the protein in Western blots. The specificity of the antisera was tested against a panel of retroviruses. Transmembrane proteins of type C retroviruses as well as type D were identified.

Low-molecular- weight Rauscher leukemia virus protein with preferential binding for single-stranded RNA and DNA.

A sensitive nitrocellulose filter assay that measures the retention of 125I single-stranded calf thymus DNA has been used to detect and purify DNA-binding proteins that retain a biological function from Rauscher murine leukemia virus. By consecutive purification on oligo (dT)- cellulose and DEAE-Bio-Gel columns and centrifugation in 10 to 30% glycerol gradients, RNA-dependent DNA polymerase has been separated from a second virion DNA-binding protein. The binding of this protein to DNA was strongly affected by NaCl concentration but showed little change in activity over a wide range of temperature or pH. After glycerol gradient purification, polyacrylamide gel electrophoresis of this protein showed one major band with a molecular weight of approximately 9,800. This protein binds about as well as to single-stranded Escherichia coli or calf thymus DNA or 70S type C viral RNA. The binding to 125I single-stranded calf thymus DNA is very efficiently inhibited by unlabeled single-stranded DNA from either E. coli or calf thymus and by 70S murine or feline viral RNA. Much larger amounts of double-stranded DNA are required to produce an equivalent percentage of inhibition. This protein, therefore, shows preferential binding to single-stranded DNA or viral RNA.

Isolation from cats of an endogenous type C virus with a novel envelope glycoprotein.

A search for variant endogenous cat viruses led to a novel isolate. Although the major envelope glycoprotein of this virus was similar in size to that of an RD-114-like virus that was coisolated, it was unrelated to RD-114 or feline leukemia virus by immunological and biological criteria. This degree of dissimilarity suggests a different evolutionary progenitor from that for the RD-114 and feline leukemia virus viral envelopes. The novel virus did, however, code for gag gene polypeptides which are closely related to RD-114 virus. Neither the novel isolate nor the RD-114-like coisolate induced foci in S+L- cat cells which restrict focus induction by RD-114 virus. This suggests that the two viruses share a common genomic target of restriction which resides outside of the env region.