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.

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.

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.

Intermittent elevation of serum urate and 24-hour urinary uric acid excretion.

OBJECTIVES: Serum urate concentrations fluctuate throughout the day, and may be subject to variation with time. However, monthly variation of urinary uric acid excretion has not been investigated. This prompted us to investigate serum urate and 24-h urinary uric acid excretion in healthy men. METHODS: Serum urate and creatinine and 24-h urinary uric acid and creatinine were measured at monthly intervals throughout a 12-month period in 12 healthy men (aged 23-61 yr) from July 2002 to June 2003. RESULTS: The mean age of the 12 healthy men was 35.3+/-10.5 yr (median 33, range 23-61), and they had mean serum urate concentration 7.1+/-1.1 mg/dl (range 4.6-10.4), mean serum creatinine 1.0+/-0.1 mg/dl (range 0.8-1.3) and mean 24-h urinary uric acid excretion 651+/-189 mg/day/1.73 m(2) (median 623, range 389-1565). Approximately 20.1 and 20.7% of the measurements displayed above normal serum urate level and daily urinary uric acid excretion of more than 800 mg, respectively. CONCLUSIONS: The data presented here demonstrate individual variations in serum urate levels and 24-h urinary uric acid excretions in healthy men with serial measurement. Transient hyperuricaemia and hyperuricosuria are more common than expected, and both transitory and monthly variations are important factors to consider when evaluating the influence of other factors upon serum urate levels and urinary uric acid excretion. Further studies are needed to confirm these results using larger populations.

Anticardiolipin antibodies in various diseases in Taiwan: a retrospective analysis.

The goals of this study are to determine the frequency of anticardiolipin antibodies (ACA) in patients with various diseases and to evaluate the clinical significance of ACA in Taiwan. We collected 690 patients from ACA laboratory records. They were divided into eight groups in order to compare ACA percentages. Positive rates of ACA in different disease groups were below 20%, except for 38.2% in autoimmune diseases with vascular thrombosis. Compared with old stroke, the ACA positivity in young stroke was not significantly different (P = 0.482). The positive percentage of lupus anticoagulant (LA) (2.86%) was lower than that of ACA (15.66%) in young stroke (P = 0.015). Among patients with pregnancy loss or prematurity, the ACA positivity in lupus patients (44.44%) was higher than without lupus (9.76%; P = 0.01). The prevalence of ACA is higher in patients with vascular thrombosis complicated by autoimmune diseases than with thrombosis alone in Taiwan. Young and old stroke do not differ in ACA positivity. Moreover, ACA is more prevalent than LA for young stroke related coagulation. The ACA positivity for pregnancy loss or prematurity is very low in Taiwan. In summary, this is the first report on the frequency of ACA and other coagulation factors in various diseases in Taiwan.

Concomitant septic and gouty arthritis--an analysis of 30 cases.

OBJECTIVES: To analyse the clinical features and outcomes of gouty patients with concomitant septic arthritis in a medical centre. METHODS: From the hospital database, we collected 30 hospitalized cases with concomitant septic arthritis and gouty arthritis from 1987 to 2001. All patients had positive bacterial culture and monosodium urate crystals in the affected joints. Medical records of the patients were analysed in detail. RESULTS: The mean age of patients was 52.8+/-12.5 yr. One-third of patients were afebrile at presentation, 30% had a normal blood leucocyte count and 10% had a synovial fluid leucocyte count less than 6000/mm3. The knee joint was the most common site of involvement, followed by the ankle, shoulder and wrist joints. Most patients had long-standing disease and subcutaneous tophi. Subcutaneous tophi rupture with secondary wound infection is the most common route of infection. Causative micro-organisms were Staphylococcus aureus (16 cases, 7 of whom were oxacillin-resistant), Streptococcus sp. (5 cases), Pediococcus sp. (1 case), and Gram-negative bacilli (9 cases). Fourteen patients received surgical debridement, among them two patients had an arthrodesis owing to severe joint destruction and one received above-knee amputation. Two patients died. One died of septic complications and the other died of acute myocardial infarction. CONCLUSIONS: Septic arthritis coexistent with gout presented a diagnostic difficulty. An early diagnosis requires a high level of suspicion. Prompt aspiration and analysis of the synovial fluid is imperative, regardless of the absence of fever or leucocytosis. Culture of the aspirated synovial fluid is warranted in gouty attack, even when it has a low white cell count or the Gram stain reveals no organisms.

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.

Potentiation of simian immunodeficiency virus (SIV)-specific CD4(+) and CD8(+) T cell responses by a DNA-SIV and NYVAC-SIV prime/boost regimen.

T cell-mediated immune responses play an important role in the containment of HIV-1 replication. Therefore, an effective vaccine against HIV-1 should be able to elicit high frequencies of virus-specific CD8(+) and CD4(+) T cells. The highly attenuated poxvirus-based vaccine candidate, NYVAC-SIV-gag-pol-env (NYVAC-SIV-gpe), has been shown to induce and/or expand SIV-specific CD4(+) and CD8(+) T cell responses in both naive and infected macaques. In this study, the immunogenicity of NYVAC-SIV-gpe alone was compared with a combination regimen where priming with an optimized DNA-SIV-gag-env vaccine candidate was followed by a NYVAC-SIV-gpe boost. In macaques immunized with the prime-boost regimen, the extent and durability of CD8(+) T cell response to an immunodominant SIV gag epitope was increased and these animals recognized a broader array of subdominant SIV epitopes in the cytolytic assay. In addition, the prime-boost regimen significantly enhanced the proliferative responses to both SIV gag and env proteins. Thus, the combination of these vaccine modalities may represent a valuable strategy in the development of a vaccine for HIV.

Impairment of Gag-specific CD8(+) T-cell function in mucosal and systemic compartments of simian immunodeficiency virus mac251- and simian-human immunodeficiency virus KU2-infected macaques.

The identification of several simian immunodeficiency virus mac251 (SIV(mac251)) cytotoxic T-lymphocyte epitopes recognized by CD8(+) T cells of infected rhesus macaques carrying the Mamu-A*01 molecule and the use of peptide-major histocompatibility complex tetrameric complexes enable the study of the frequency, breadth, functionality, and distribution of virus-specific CD8(+) T cells in the body. To begin to address these issues, we have performed a pilot study to measure the virus-specific CD8(+) and CD4(+) T-cell response in the blood, lymph nodes, spleen, and gastrointestinal lymphoid tissues of eight Mamu-A*01-positive macaques, six of those infected with SIV(mac251) and two infected with the pathogenic simian-human immunodeficiency virus KU2. We focused on the analysis of the response to peptide p11C, C-M (Gag 181), since it was predominant in most tissues of all macaques. Five macaques restricted viral replication effectively, whereas the remaining three failed to control viremia and experienced a progressive loss of CD4(+) T cells. The frequency of the Gag 181 (p11C, C-->M) immunodominant response varied among different tissues of the same animal and in the same tissues from different animals. We found that the functionality of this virus-specific CD8(+) T-cell population could not be assumed based on the ability to specifically bind to the Gag 181 tetramer, particularly in the mucosal tissues of some of the macaques infected by SIV(mac251) that were progressing to disease. Overall, the functionality of CD8(+) tetramer-binding T cells in tissues assessed by either measurement of cytolytic activity or the ability of these cells to produce gamma interferon or tumor necrosis factor alpha was low and was even lower in the mucosal tissue than in blood or spleen of some SIV(mac251)-infected animals that failed to control viremia. The data obtained in this pilot study lead to the hypothesis that disease progression may be associated with loss of virus-specific CD8(+) T-cell function.

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.

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.

[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.

A human vascular endothelial cell model to study angiogenesis and tumorigenesis.

Endothelial cell biology has recently been the subject of considerable interest in thrombosis and cancer research. However, the successful establishment of immortalized human endothelial cells which retain differentiated cell characteristics has been rare. We have successfully established immortalized human umbilical vein endothelial cells (HUVECs) by human papilloma virus (HPV)-16 E6-E7. HPV-16 E6, E7 and E6-E7 were successfully introduced into HUVEC cells. Both E6 and E7 cultures had an extended lifespan but eventually underwent senescence. E6-E7 cultures 4-5-2G, however, acquired an indefinite lifespan in culture but did not undergo malignant conversion. Telomerase activity was not detected in either E6 or E7 cultures; however, telomerase was detected in E6-E7 4-5-2G cells. The cells exhibited a 'cobblestone' morphology and developed a capillary-like tube structure upon reaching confluence. The 4-5-2G line expressed Factor VIII related antigen and took up DiI-Ac-LDL as markers of endothelial origin. The line expressed integrin subunits (alpha(v)beta3, alph(v)beta5, beta1, alpha2, alpha3, beta4 and alpha6) consistent with an endothelial origin. The higher passage of 4-5-2G line showed a similar intensity of integrin immunostaining to that of primary HUVECS. Subsequent infection of these immortal cells with the Kirsten murine sarcoma virus which contains an activated K-ras oncogene induced morphological transformation that led to the acquisition of invasion capability and neoplastic properties. Telomerase was also detected in the tumorigenic v-Ki-ras transformed cell line. These cell lines should be useful for studies of the molecular mechanisms underlying normal and neoplastic endothelial cell proliferation and migration, and might also provide an in vitro model for development of pharmacologic and gene therapy for cardiovascular thrombosis and cancer.

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.

Refocusing neutralizing antibody response by targeted dampening of an immunodominant epitope.

Immunodominant epitopes are known to suppress a primary immune response to other antigenic determinants by a number of mechanisms. Many pathogens have used this strategy to subvert the immune response and may be a mechanism responsible for limited vaccine efficacies. HIV-1 vaccine efficacy appears to be complicated similarly by a limited, immunodominant, isolate-restricted immune response generally directed toward determinants in the third variable domain (V3) of the major envelope glycoprotein, gp120. To overcome this problem, we have investigated an approach based on masking the V3 domain through addition of N-linked carbohydrate and reduction in net positive charge. N-linked modified gp120s were expressed by recombinant vaccinia virus and used to immunize guinea pigs by infection and protein boosting. This modification resulted in variable site-specific glycosylation and antigenic dampening, without loss of gp120/CD4 binding or virus neutralization. Most importantly, V3 epitope dampening shifted the dominant type-specific neutralizing Ab response away from V3 to an epitope in the first variable domain (V1) of gp120. Interestingly, in the presence of V3 dampening V1 changes from an immunodominant non-neutralizing epitope to a primary neutralizing epitope with broader neutralizing properties. In addition, Ab responses were also observed to conserved domains in C1 and C5. These results suggest that selective epitope dampening can lead to qualitative shifts in the immune response resulting in second order neutralizing responses that may prove useful in the fine manipulation of the immune response and in the development of more broadly protective vaccines and therapeutic strategies.

Preliminary in vitro growth cycle and transmission studies of HIV-1 in an autologous primary cell assay of blood-derived macrophages and peripheral blood mononuclear cells.

Recent interest focused on the dynamics of HIV-1 replication in primary monocytes/macrophages and T-lymphocytes of the immune system, as well as the standardization of virological and immunological in vitro assays with primary isolates, provided the impetus for these studies. These types of studies have never been performed as they would occur in vivo, i.e., where the envelope of the virus and cell membranes of the two cell types of the same host origin. Therefore, the biological and physicochemical properties of an uncloned, primary dual-tropic isolate HIV-1ADA during the initial lag, log, and stationary phases of viral replication were studied in an autologous donor cell assay in peripheral blood mononuclear cells (PBMC) and blood monocyte-derived macrophages (MDM). Similar total numbers (10(9) virus particles/ml) were produced by both cell types during the stationary period. On a per cell per day basis, during peak stationary periods, 0.92 x 10(3) virions/day for MDMs and 5.31 x 10(3) virions/day for PBMCs were produced. Interestingly, virus replicating from MDMs during the log-growth phase demonstrated the greatest infectious fraction which was 3 logs greater than virus replicating in PBMCs. Despite constant virus particle production in MDMs, the infectious fraction was found to fall 3 to 4 logs over a 10-day period. Due to an infectious fraction less than 1 (0.053 infectious unit/cell/24 hr), virus spread in PBMCs during the rapid log phase could only have occurred by cell-to-cell contact, whereas in MDMs with an infectious fraction of about one infectious particle (approximately 1/cell/24 hr), cell-free transmission could account for the observed results. Most of the MDMs (> 90%) became productively infected, whereas only 5-10% of the total PBMCs were found replicating virus. The period of peak stationary virus production (i.e., stationary phase) was at minimum 4 to 5 times longer in MDMs than PBMCs. Whereas the majority of p24, RT, and gp 120 found to be associated with MDM-derived virions, no increased dissociation of these components was observed in PBMC-derived virions. The virion-associated gp 120 was 3 to 4 times more stable on both PBMC- and MDM-derived virus (> 96 hr) and present at 10-25 times the concentration per virion than that observed for a T-cell-line-adapted laboratory strain of HIV-1 replicating in T-cell lines. These in vitro results suggest that important differences exist between MDMs and PBMCs with regard to the viral dynamics of infection and replication which should provide for a qualitative and quantitative basis to estimate virus replication on a per-cell basis for other known cellular targets of HIV-1. Studying the multiple biophysicochemical characteristics and viral replication dynamics as described herein provides an autologous in vitro model of additional quantifiable parameters for analysis and understanding of virus/host factor(s) and/or antivirals which influence them.

Selective oxidative modification and affinity cleavage of pigeon liver malic enzyme by the Cu(2+)-ascorbate system.

Pigeon liver malic enzyme was rapidly inactivated by micromolar concentration of Fe2+ in the presence of ascorbate at neutral pH. The inactivated enzyme was subsequently cleaved by the Fe(2+)-ascorbate system at the chemical bond between Asp258 and Ile259 (Wei, C.H., Chou, W.Y., Huang, S.M., Lin, C.C., and Chang, G.G. (1994) Biochemistry, 33, 7931-7936), which was confirmed by site-specific mutagenesis (Wei, C.H., Chou, W.Y., and Chang, G.G. (1995) Biochemistry 34, 7949-7954). In the present study, at neutral pH, Cu2+ was found to be more reactive in the oxidative modification of malic enzyme and the enzyme was cleaved in a similar manner as Fe2+ did. At acidic pH, however, Fe2+ was found to be ineffective in oxidative modification of the enzyme. Nevertheless, Cu2+ still caused enzyme inactivation and cleaved the enzyme at Asp141-Gly142, Asp194-Pro195, or Asp464-Asp465. Mn2+ and L-malate synergistically protect the enzyme from Cu2+ inactivation at acidic pH. Cu2+ is also a competitive inhibitor versus Mn2+ in the malic enzyme-catalyzed reaction with Ki value 70.3 +/- 5.8 microM. The above results indicated that, in addition to the previously determined Asp258 at neutral pH, Asp141, Asp194, and Asp464 are also the coordination sites for the metal binding of malic enzyme. We suggest that the mechanism of affinity modification and cleavage of malic enzyme by the Cu(2+)-ascorbate system proceed in the following sequence. First, Cu2+ binds with the enzyme at the Mn2+ binding site and reduces to Cu+ by ascorbate. Next, the local oxygen molecules are reduced by Cu+, thereby generating superoxide or other reactive free radicals. These radicals interact with the susceptible essential amino acid residues at the metal-binding site, ultimately causing enzyme inactivation. Finally, the modified enzyme is cleaved into several peptide fragments, allowing the identification of metal site of the enzyme. The pH-dependent different specificities of metal-catalyzed oxidation system may be generally applicable for other enzymes or proteins.