Peripheral-derived regulatory T cells contribute to tumor-mediated immune suppression in a nonredundant manner.

Identifying tumor-mediated mechanisms that impair immunity is instrumental for the design of new cancer therapies. Regulatory T cells (Tregs) are a key component of cancer-derived immune suppression; however, these lymphocytes are necessary to prevent systemic autoimmunity in mice and humans, and thus, direct targeting of Tregs is not a clinical option for cancer patients. We have previously demonstrated that excising transcription factor Kruppel-like factor 2 (Klf2) within the T cell lineage blocks the generation of peripheral-derived Tregs (pTregs) without impairing production of thymic-derived Tregs. Using this mouse model, we have now demonstrated that eliminating pTregs is sufficient to delay/prevent tumor malignancy without causing autoimmunity. Cancer-bearing mice that expressed KLF2 converted tumor-specific CD4+ T cells into pTregs, which accumulated in secondary lymphoid organs and impaired further T cell effector activity. In contrast, pTreg-deficient mice retained cancer-specific immunity, including improved T cell infiltration into "cold" tumors, reduced T cell exhaustion in tumor beds, restricted generation of tumor-associated myeloid-derived suppressor cells, and the continued production of circulating effector T cells that arose in a cancer-dependent manner. Results indicate that tumor-specific pTregs are critical for early stages of cancer progression and blocking the generation of these inhibitory lymphocytes safely delays/prevents malignancy in preclinical models of melanoma and prostate cancer.

Dendrimer-enabled transformation of Chlamydia trachomatis.

Lack of a system for genetic manipulation of Chlamydia trachomatis has been a key challenge to advancing understanding the molecular genetic basis of virulence for this bacterial pathogen. We developed a non-viral, dendrimer-enabled system for transformation of this organism and used it to characterize the effects of inserting the common 7.5 kbp chlamydial plasmid into strain L2(25667R), a C. trachomatis isolate lacking it. The plasmid was cloned in pUC19 and the clone complexed to polyamidoamine dendrimers, producing ∼83 nm spherical particles. Nearly confluent McCoy cell cultures were infected with L2(25667R) and reference strain L2(434). At 16 h post-infection, medium was replaced with dendrimer-plasmid complexes in medium lacking additives (L2(25667R)) or with additive-free medium alone (L2(434)). Three h later complexes/buffer were removed, and medium was replaced; cultures were harvested at various times post-transformation for analyses. Real time PCR and RT-PCR of nucleic acids from transformed cultures demonstrated plasmid replication and gene expression. A previous report indicated that one or more plasmid-encoded product govern(s) transcription of the glycogen synthase gene (glgA) in standard strains. In L2(25667R) the gene is not expressed, but transformants of that strain given the cloned chlamydial plasmid increase glgA expression, as does L2(434). The cloned plasmid is retained, replicated, and expressed in transformants over at least 5 passages, and GFP is expressed when transformed into growing L2(25667R). This transformation system will allow study of chlamydial gene function in pathogenesis.

Dendrimer-enabled DNA delivery and transformation of Chlamydia pneumoniae.

The chlamydiae are important human pathogens. Lack of a genetic manipulation system has impeded understanding of the molecular bases of virulence for these bacteria. We developed a dendrimer-enabled system for transformation of chlamydiae and used it to characterize the effects of inserting the C. trachomatis plasmid into C. pneumoniae, which lacks any plasmids. The plasmid was cloned into modified yeast vector pEG(KG) and the clone complexed to polyamidoamine dendrimers, producing 50-100 nm spherical particles. HEp-2 cell cultures were infected with C. pneumoniae strain AR-39. Twenty-four hours later, medium was replaced for 3 hours with dendrimer-plasmid complexes, then removed and the medium replaced. Cultures were harvested at various times post-transformation. Real-time PCR and RT-PCR of nucleic acids from transformed cultures demonstrated plasmid replication and gene expression. The cloned plasmid was replicated and expressed in transformants over 5 passages. This system will allow study of chlamydial gene function, allowing development of novel dendrimer-based therapies. FROM THE CLINICAL EDITOR: This team of investigators developed a dendrimer-enabled system for transformation of chlamydiae and successfully utilized it to characterize the effects of inserting the C. trachomatis plasmid into C. pneumonia. This system will allow study of chlamydial gene function, allowing development of novel dendrimer-based therapies.

Dendrimer-enabled modulation of gene expression in Chlamydia trachomatis.

The obligate intracellular bacterium Chlamydia trachomatis is an important human pathogen. The genome of this organism is small but encodes many genes of currently unknown function that are thought to be involved in virulence. Lack of a system for genetic manipulation has been a key challenge to advancing the understanding of molecular genetics underlying virulence for this bacterium. We developed a dendrimer-enabled system for transformation of C. trachomatis, and used it to demonstrate the efficient and highly specific knockdown of transcript levels from targeted genes. Antisense, sense, and other control oligonucleotides targeting two sets of duplicated genes on the chlamydial chromosome were designed, commercially synthesized, and complexed with generation-4 polyamidoamine (PAMAM) dendrimers. The complexes were given to HEp-2 cell cultures infected for 16 h with C. trachomatis serovar K and then removed three hours later. Infected cultures were harvested 6 h after pulsing, and DNA and RNA/cDNA were prepared for assessment of transcript levels compared to those for the same genes in infected cultures, without dendrimer complexation. In all cases, the targeted gene complexed to dendrimer, but not its duplicate, showed up to 90% transcript attenuation. The duration of attenuation can be extended by repeated pulsing, and in some cases transcript levels from multiple genes can be attenuated in the same organism. This system will allow study of chlamydial gene function in pathogenesis, leading to more effective therapies to treat Chlamydia-induced diseases in a targeted manner.

PAMAM dendrimer-azithromycin conjugate nanodevices for the treatment of Chlamydia trachomatis infections.

Chlamydia trachomatis is an important bacterial pathogen known to be etiological in genital infections, as well as several serious disease sequelae, including inflammatory arthritis. Chlamydiae can persist in infection, making treatment with antibiotics such as azithromycin (AZ) a challenge. The authors explore the use of neutral generation-4 polyamidoamine (PAMAM) dendrimers as intracellular drug-delivery vehicles into chlamydial inclusions. Azithromycin was successfully conjugated with the dendrimers, and the conjugate (D-AZ) released ≈ 90% of the drug over 16 hours. The conjugate readily entered both the Chlamydia-infected HEp-2 cells and the chlamydial inclusions. The conjugate was significantly better than free drug in preventing productive infections in the cells when added at the time of infection, and better in reducing the size and number of inclusions when added either 24 hours or 48 hours post infection. These studies show that dendrimers can deliver drugs efficiently to growing intracellular C. trachomatis, even if the organism is in the persistent form. FROM THE CLINICAL EDITOR: In this report, the use of polyamidoamine dendrimers as intracellular drug-delivery vehicles into chlamydial inclusions is investigated. This method results in efficient intracellular delivery of azithromycin to address chlamydia infection.

Up-regulation of a cellular protein at the translational level by a retrovirus.

Mink cell focus-forming (MCF) murine leukemia viruses (MLVs) are the etiologic agent of thymic lymphoma in mice. We have observed previously that superinfection by MCF13 MLV of certain cell types, such as preleukemic thymic lymphocytes and cultured mink epithelial cells, results in the accumulation of the viral envelope precursor polyprotein, leading to the induction of endoplasmic reticulum (ER) stress. In this study, we demonstrate that the induction of ER stress by MCF13 MLV infection results in an increase in the phosphorylation of the alpha-subunit of eukaryotic initiation factor 2. In cells in which this occurs, we have detected an up-regulation of the cellular inhibitor of apoptosis protein 1 (c-IAP1). The results of real-time RT-PCR quantification of message levels and protein turnover assays indicate that up-regulation of c-IAP1 occurs at the translational level. Elevation of c-IAP1 levels at a posttranscriptional step was detectable in MCF13 MLV-induced thymic lymphomas and chronically infected mink epithelial cells. The ability of a simple retrovirus to regulate cellular gene expression at the translational level may be an important mechanism that contributes to pathogenesis.

The pathogenic role of Chlamydia in spondyloarthritis.

PURPOSE OF REVIEW: Topics relating to the spondyloarthropathies have been reviewed recently, but the detailed roles of Chlamydia trachomatis and C. pneumoniae in induction of spondyloarthritis have not been discussed. This review focuses on new information regarding how these pathogens elicit joint disease, with emphasis on C. trachomatis in its role in Chlamydia-induced reactive arthritis. RECENT FINDINGS: Molecular methods continue to provide insights into the molecular genetic and cell biologic basis for chlamydial pathogenesis. For chlamydiae, residence in the synovium in patients with acute or chronic Chlamydia-induced arthritis involves organisms in an unusual infection state designated persistence. The profiles of overall metabolism and gene expression characteristic of chlamydial persistence have been assessed and unusual aspects noted, including transcriptional attenuation of one hsp60 paralog and upregulation of expression for another. Strain determinations have demonstrated that genital serotypes of C. trachomatis are not present in the joint; rather, inflammation at that site is elicited by ocular serotypes of the organism. This indicates that much remains to be learned concerning the biology of chlamydial dissemination from the urogenital tract. Analyses of undifferentiated spondyloarthritis continue to suggest that chlamydiae, and perhaps other pathogens function in the etiology of the disease. Progress has been made in developing effective treatment for patients with Chlamydia-induced arthritis. SUMMARY: Molecular genetic analyses regarding the role of chlamydiae in induction of inflammatory arthritis have increased our detailed understanding of the pathogenic mechanisms utilized by these organisms in the joint. Importantly, progress has been made in developing effective therapies for treatment of Chlamydia-induced arthritis.

Patients with Chlamydia-associated arthritis have ocular (trachoma), not genital, serovars of C. trachomatis in synovial tissue.

Some individuals with a genital Chlamydia trachomatis infection develop inflammatory arthritis, but it is unknown whether particular chlamydial serovar(s) engender the disease more often than others. We defined serovar in synovial tissues from arthritis patients infected with this organism. DNA from synovial biopsies of 36 patients with PCR-confirmed synovial C. trachomatis was analyzed. Diagnoses included reactive arthritis, undifferentiated oligoarthritis, rheumatoid arthritis, and osteoarthritis. The chlamydial omp1 and trpA genes were amplified, cloned, and 10 or more clones from each sample were sequenced. The cytotoxin locus also was analyzed. omp1 sequences showed 2 patients having only C. trachomatis A serovar, 1 with only B, and 33 having only C, all ocular serovars. Analyses of trpA and the cytotoxin locus uniformly displayed standard ocular serovar characteristics for each patient. Identification of ocular chlamydial serovars in the synovia of arthritis patients is unexpected. These observations suggest that urogenital chlamydial infections, while consisting primarily of organisms of genital serovars, include some of ocular serovar(s). They further suggest that during such infections unknown selection pressures favor establishment of the latter in the synovium to the exclusion of genital serovar chlamydiae.

Initial characterization of Chlamydophila (Chlamydia) pneumoniae cultured from the late-onset Alzheimer brain.

Previous studies from this laboratory provided evidence that the intracellular bacterial pathogen Chlamydophila (Chlamydia) pneumoniae is present in the late-onset Alzheimer's disease (AD) brain. Here we report culture of the organism from two AD brain samples, each of which originated from a different geographic region of North America. Culturable organisms were detectable after one and two passages in HEp-2 cells for the two samples. Both isolates, designated Tor-1 and Phi-1, were demonstrated to be authentic C. pneumoniae using PCR assays targeting the C. pneumoniae-specific genes Cpn0695, Cpn1046, and tyrP. Assessment of inclusion morphology and quantitation of infectious yields in epithelial (HEp-2), astrocytic (U-87 MG), and microglial (CHME-5) cell lines demonstrated an active, rather than a persistent, growth phenotype for both isolates in all host cell types. Sequencing of the omp1 gene from each isolate, and directly from DNA prepared from several additional AD brain tissue samples PCR-positive for C. pneumoniae, revealed genetically diverse chlamydial populations. Both brain isolates carry several copies of the tyrP gene, a triple copy in Tor-1, and predominantly a triple copy in Phi-1 with a minor population component having a double copy. This observation indicated that the brain isolates are more closely related to respiratory than to vascular/atheroma strains of C. pneumoniae.

Lack of evidence for bacterial infections in skin in patients with systemic sclerosis.

BACKGROUND: In some patients with systemic sclerosis (SSc), persistent bacterial infection involving dermal microvascular endothelial cells may result in endothelial injury, leading to the obliterative microvasculopathy typical of the disease. Alternatively, in some patients with SSc persistent bacterial infection involving activated dermal fibroblasts or other cells found in scleroderma skin might result in the fibrosing features of this disease. In this study, we investigated bacterial infection in skin in patients with SSc. METHODS: Chlamydiae of many species are known to undergo persistent infection. Highly sensitive and specific PCR assays targeting chromosomal DNA sequences from C. trachomatis and C. pneumoniae were used to screen skin biopsy samples from each of 18 patients and 26 control individuals. Additional screening was performed using a highly sensitive "pan-bacteria" PCR screening system. RESULTS: All patient and control samples proved to be PCR-negative for both chlamydial species. Similarly, all patient and control samples were PCR-negative when the broad range pan-bacteria assay system was used. CONCLUSION: Although some caveats apply, the data presented here do not support the contention that persistent bacterial infections play an important role in the pathogenesis of SSc.

Chlamydophila pneumoniae and the etiology of late-onset Alzheimer's disease.

Sporadic, late-onset Alzheimer's disease (LOAD) is a non-familial, progressive neurodegenerative disease that is now the most common and severe form of dementia in the elderly. That dementia is a direct result of neuronal damage and loss associated with accumulations of abnormal protein deposits in the brain. Great strides have been made in the past 20 years with regard to understanding the pathological entities that arise in the AD brain, both for familial AD ( approximately 5% of all cases) and LOAD ( approximately 95% of all cases). The neuropathology observed includes: neuritic senile plaques (NSPs), neurofibrillary tangles (NFTs), neuropil threads (NPs), and often deposits of cerebrovascular amyloid. Genetic, biochemical, and immunological analyses have provided a relatively detailed knowledge of these entities, but our understanding of the "trigger" events leading to the many cascades resulting in this pathology and neurodegeneration is still quite limited. For this reason, the etiology of AD, in particular LOAD, has remained elusive. However, a number of recent and ongoing studies have implicated infection in the etiology and pathogenesis of LOAD. This review focuses specifically on infection with Chlamydophila (Chlamydia) pneumoniae in LOAD and how this infection may function as a "trigger or initiator" in the pathogenesis of this disease.

Autoimmune Th2-mediated dacryoadenitis in MRL/MpJ mice becomes Th1-mediated in IL-4 deficient MRL/MpJ mice.

PURPOSE: MRL/MpJ mice of substrains MRL/MpJ-fas(+)/fas(+) (MRL/+) and MRL/MpJ-fas(lpr)/fas(lpr) (MRL/lpr) spontaneously develop autoimmune dacryoadenitis and sialadenitis and are a model for the human disorder Sjögren syndrome. The dacryoadenitis in both substrains appears to be Th2 in nature, with little IFN-gamma and substantial IL-4 at the site of lacrimal gland inflammation. METHODS: MRL/MpJ mice with a defective IL-4 gene-both MRL/+-IL-4(tm)/IL-4(tm) (MRL/+/IL-4(tm)) and MRL/lpr-IL-4(tm)/IL-4(tm) (MRL/lpr-IL-4(tm))-that resulted in a loss of IL-4 production were bred and evaluated for dacryoadenitis. RESULTS: MRL/+/IL-4(tm) and MRL/lpr/IL-4(tm) mice developed dacryoadenitis of similar onset, appearance, and severity as found in MRL/MpJ mice with an intact IL-4 gene. Immunohistochemistry examination revealed a substantially greater number of inflammatory cells staining for IFN-gamma than for IL-13 in the dacryoadenitis of IL-4-deficient MRL/MpJ mice (MRL/+/IL-4(tm), 66% vs. 0.8%, P = 0.001; MRL/lpr/IL-4(tm), 67% vs. 1.2%, P = 0.002). Real-time PCR demonstrated greater amounts of IFN-gamma than IL-13 mRNA relative transcripts in lacrimal glands of MRL/lpr/IL-4(tm) mice (mean difference, 28.6; P = 0.035). Greater CD86 (B7-2) than CD80 (B7-1) expression was present in MRL/+/IL-4(tm) mice (11% vs. 3%, P = 0.003) and MRL/lpr/IL-4(tm) mice (10% vs. 3%, P = 0.002). CONCLUSIONS: These results suggest that a Th2 autoimmune process can be converted to a Th1 process in the absence of IL-4.

Chlamydophila (Chlamydia) pneumoniae in the Alzheimer's brain.

We assessed the presence and characteristics of the intracellular pathogen Chlamydophila (Chlamydia) pneumoniae in brain-tissue samples from 25 patients with late-onset Alzheimer's disease (AD) and 27 non-AD control individuals. 20/27 AD patients, but only 3/27 controls, were PCR-positive in multiple assays targetting the Cpn1046 and Cpn0695 genes. Culture of the organism from brain-tissue homogenate from one AD patient, and assessment of various chlamydial transcripts in RNA preparations from several patients, demonstrated that the organisms were viable and metabolically active in those samples. Immunohistochemical analyses showed that astrocytes, microglia, and neurons all served as host cells for C. pneumoniae in the AD brain, and that infected cells were found in close proximity to both neuritic senile plaques and neurofibrillary tangles in the AD brain. These observations confirm and significantly extend our earlier study suggesting that this unusual pathogen may play a role in the neuropathogenesis characteristic of AD.

Chlamydophila (Chlamydia) pneumoniae infection of human astrocytes and microglia in culture displays an active, rather than a persistent, phenotype.

BACKGROUND: The intracellular pathogen Chlamydia pneumoniae can cause persistent infections during which its morphologic, molecular, and pathogenic characteristics differ importantly from those of active infection. This bacterium was identified within astrocytes and microglia in the brain of late-onset Alzheimer disease patients. We investigated whether infection of these two host cell types displays an active or persistent growth phenotype. METHODS: The human astrocytoma and microglioma cell lines U-87 MG and CHME-5 (respectively) and the human epithelial cell line HEp-2 were infected by the standard method with C pneumoniae strain AR-39. Cultures were harvested at 24, 48, and 72 hours postinfection and subjected to analysis of inclusion morphology. DNA and RNA were prepared from portions of each infected culture sample and analyzed for relative chromosome accumulation and presence or absence of several specific bacterial mRNAs. RESULTS: Astrocytes and microglial cells infected in vitro with C pneumoniae displayed inclusions that were indistinguishable from those characteristic of active infection of the standard HEp-2 host cell line. Real time polymerase chain reaction (PCR) showed that the relative accumulation of chlamydial chromosome over time during infection of these two cell lines also was virtually identical to that in actively infected HEp-2 cells. Reverse transcriptase PCR (RT-PCR) analyses showed that mRNA from ftsK, pyk, and other chlamydial genes whose expression is abrogated during persistent infection were easily identifiable in infected CHME-5 and U-87 MG cells. CONCLUSIONS: In cultured human astrocytes and microglia, C pneumoniae displays an active, not a persistent, growth phenotype. This indicates normal passage through the developmental cycle with its probable concomitant destruction by lysis of some portion of host cells at the termination of that cycle.

Chlamydia trachomatis genes whose products are related to energy metabolism are expressed differentially in active vs. persistent infection.

The Chlamydia trachomatis genome encodes glycolysis and pentose phosphate pathway enzymes, two ATP/ADP exchange proteins, and other energy transduction-related components. We asked if and when chlamydial genes specifying products related to energy transduction are expressed during active vs. persistent infection in in vitro models and in synovia from Chlamydia-associated arthritis patients. Hep-2 cells infected with K serovar were harvested from 0-48 h post-infection (active infection). Human monocytes identically infected were harvested at 1, 2, 3, 5 days post-infection (persistent). RNA from each preparation and from synovial samples PCR-positive/-negative for Chlamydia DNA was subjected to RT-PCR targeting (a) chlamydial primary rRNA transcripts and adt1 mRNA, (b) chlamydial mRNA encoding enzymes of the glycolysis (pyk, gap, pgk) and pentose phosphate (gnd, tal) pathways, the TCA cycle (mdhC, fumC), electron transport system (cydA, cydB), and sigma factors (rpoD, rpsD, rpoN). Primary rRNA transcripts and adt1 mRNA were present in each infected preparation and patient sample; controls were negative for chlamydial RNA. In infected Hep-2 cells, all energy transduction-related genes were expressed by approximately 11 h post-infection. In monocytes, pyk, gap, pgk, gnd, tal, cydA mRNA were present in 1-2-day-infected cells but absent at 3 days and after; cydB, mdhC, fumC were expressed through 5 days post-infection. RT-PCR targeting mRNA from sigma factor genes indicated that lack of these gene products cannot explain selective transcriptional down-regulation during persistence. Analyses of RNA from synovial tissues mirrored those from the monocyte system. These data suggest that in the first phase of active chlamydial infection, ADP/ATP exchange provides energy required for metabolism; in active growth, glycolysis supplements host ATP. In persistence host, rather than bacterially produced, ATP is the primary energy source. Metabolic rate in persistent C. trachomatis is lower than in actively growing cells, as judged from assays for relative chlamydial primary rRNA transcript levels in persistent vs. actively growing cells.

Chemokines in autoimmune lacrimal gland disease in MRL/MpJ mice.

PURPOSE: MRL/MpJ-fas+/fas+ (MRL/+) and MRL/MpJ-fas(lpr)/fas(lpr) (MRL/lpr) mice undergo spontaneous development of inflammation of the lacrimal and salivary glands, similar to that in the human disorder Sjögren's syndrome. Previous work has shown that these lesions appear to be largely T helper (Th)-2-driven, as evidenced by the substantially greater expression of IL-4 than interferon-gamma. The relative contributions of selected chemokines associated with Th1 and Th2 immune responses were assessed. METHODS: Lacrimal glands from MRL/+ and MRL/lpr mice, at ages 1.5 through 9 months were evaluated by immunohistochemistry for the chemokines monocyte chemoattractant protein (MCP)-1 (also known as chemokine ligand [CCL]-2), MCP-5 (CCL12), thymus activation regulated chemokine (TARC; or CCL17), and macrophage-derived chemokine (MDC; or CCL22). Additional lacrimal glands were tested by real-time RT-PCR for chemokines MCP-1 and -5, which are associated with Th2 and Th1 responses, respectively. RESULTS: By immunohistochemistry a significantly greater proportion of mononuclear inflammatory cells in the lacrimal gland lesions stained for MCP-1 (29%-48% depending on age) compared with MCP-5 (1%-3% depending on age) both in MRL/+ (mean difference 34.2%, P < 0.001) and MRL/lpr (mean difference 33.6%, P < 0.001) substrains. Real-time RT-PCR studies showed higher transcript levels of MCP-1 compared with MCP-5, in both MRL/+ (median difference, 37.3; P < 0.0001) and MRL/lpr (median difference, 77.1; P < 0.0001) mice. Relative transcripts of MCP-1 increased with age in both MRL/+ mice (P = 0.02) and MRL/lpr mice (P = 0.03). Staining for TARC was present on lacrimal gland ductular cells but not on the infiltrating lymphocytes, and staining for MDC was present on scattered individual cells throughout the lacrimal gland, but not on infiltrating lymphocytes. CONCLUSIONS: The predominant expression of a Th2-associated chemokine in the lacrimal gland lesions in this murine model of Sjögren's syndrome may contribute to the predominantly Th2-type lymphoid infiltrate in these tissues.

Inflammatory mediators in autoimmune lacrimal gland disease in MRL/Mpj mice.

PURPOSE: MRL/MpJ-fas(+)/fas(+) (MRL/+) and MRL/MpJ-fas(lpr)/fas(lpr) (MRL/lpr) mice are congenic substrains of mice that have spontaneously developing lacrimal and salivary gland inflammation and are models for the human disorder Sjögren's syndrome. Nitric oxide (NO) and tumor necrosis factor (TNF)-alpha are proinflammatory and potential mediators of tissue damage. The presence of the inducible form of nitric oxide synthase (iNOS), which catalyzes the production of NO, and the presence TNF-alpha in the lacrimal glands of MRL/MpJ mice were assessed. METHODS: Lacrimal glands from MRL/+ and MRL/lpr mice, at ages 1 through 9 months, were evaluated by real-time RT-PCR for iNOS and TNF-alpha mRNA and by immunohistochemistry for the presence of iNOS and of TNF-alpha. Age-matched BALB/c lacrimal glands were used as the control. RESULTS: By quantitative real-time PCR (qPCR), mRNA for iNOS was detected in the lacrimal glands in significantly greater amounts in both MRL/+ (median, normalized to 18S rRNA, 2.90; P < 0.0003) and MRL/lpr mice (median 6.84, P < 0.001) than in BALB/c mice (median 0.34). By qPCR, mRNA for TNF-alpha in the lacrimal glands was detected in significantly greater amounts in aged MRL/+ mice than in BALB/c mice (median, normalized to actin, 221.8 vs. 77.8, P = 0.011) and in MRL/lpr mice than in BALB/c mice (median 136.7 vs. 72.5, P = 0.001). Immunohistochemistry demonstrated both iNOS and TNF-alpha in scattered mononuclear cells throughout the lacrimal glands and in mononuclear cells at the junction of the focal inflammatory infiltrates and normal acinar tissue in both MRL/+ and MRL/lpr mice. CONCLUSIONS: As demonstrated by the greater presence of iNOS and TNF-alpha in the lacrimal glands of MRL/MpJ mice than in control glands, both NO and TNF-alpha are potential mediators of lacrimal gland damage in these murine models of Sjögren's syndrome.

Chlamydia trachomatis is present and metabolically active during the remitting phase in synovial tissues from patients with chronic Chlamydia-induced reactive arthritis.

BACKGROUND: Patients with chronic Chlamydia-induced reactive arthritis (ReA) often show a remitting-relapsing disease phenotype. Some information regarding bacterial and host responses to one another during active disease is available but no information for quiescence. This article presents the first molecular genetic insight into the behavior of bacterium and host during remitting ReA. METHODS: Synovial biopsies were procured from the knees of 4 patients with quiescent ReA by the Parker-Pearson technique. Nucleic acids prepared from them were analyzed by real-time polymerase chain reaction (PCR) and reverse transcription-PCR, and results were compared with data averaged from the knee synovial tissue samples of 10 patients with active ReA. RESULTS: Real-time PCR indicated that bacterial load in remitting samples was approximately 20% of that in active disease samples. Transcripts from the p60-encoding gene were equal to or higher than those seen in active disease. Messenger RNAs (mRNAs) from the paralog p60-encoding genes were equal to or lower than those of active disease. Host mRNAs encoding interleukin-10, tumor necrosis factor-α and interferon-γ were 4-fold lower than those in active disease samples, whereas monocyte chemotactic protein 1 and regulated upon activation, normal t-cell expressed, and secreted mRNA levels were equal to or higher. CONCLUSIONS: Bacterial load in synovial tissue of patients with remitting disease is lower than that of active disease, but mRNAs encoding proinflammatory proteins are equal to or higher than those of active disease. Transcription in the host is attenuated for cytokines and chemokines. These initial results demonstrate that organism is present and metabolically active in synovium during the remitting phase of chronic Chlamydia-induced ReA and that the genetic events characterizing quiescence are complex.

Attack rate of Chlamydia-induced reactive arthritis and effect of the CCR5-Delta-32 mutation: a prospective analysis.

OBJECTIVE: Factors that predispose patients to Chlamydia-induced reactive arthritis (CiReA) are poorly defined. Data indirectly suggest chemokine receptor-5 (CCR5)-delta-32 mutation might play a role in CiReA. We investigated the attack rate of CiReA and we hypothesized that the CCR5-delta-32 allele may modulate disease susceptibility. METHODS: Patients who tested positive for Chlamydia trachomatis after either (1) symptoms of an acute venereal disease or (2) sexual contact with an individual known to be positive for the same organism were followed in a prospective fashion. All patients were contacted at Week 6 after their acute infection and queried for symptoms of CiReA. Patients who had new-onset symptoms suggestive of CiReA were followed at Weeks 12, 26, and 52. All subjects were tested for CCR5-delta-32 mutation. RESULTS: A total of 365 study participants were enrolled, with average age 24.4 years, 201 men (55%) and 164 women (45%). We followed up with 149 patients (41%) at Week 6. Twelve of 149 participants (8.1%) had symptoms suggestive of CiReA at Week 6. None of these 12 patients was positive for the CCR5-delta-32 mutation. Of the 12 patients that had symptoms at Week 6, we were able to follow up with 7 through Week 52. All 7 had complete resolution of their symptoms by Week 26. Overall, 25/365 (6.8%) subjects were positive for the CCR5-delta-32 mutation. CONCLUSION: The attack rate of CiReA in our study was higher than previously reported, but the CCR5-delta-32 mutation does not seem to play a role in CiReA disease susceptibility.

The molecular basis for disease phenotype in chronic Chlamydia-induced arthritis.

Genital Chlamydia trachomatis infections can elicit an inflammatory arthritis in some individuals, and recent surprising studies have demonstrated that only ocular (trachoma) strains, not genital strains, of the organism are present in the synovial tissues of patients with the disease. This observation suggests an explanation for the small proportion of genitally-infected patients who develop Chlamydia-induced arthritis. Other recent studies have begun to identify the specific chlamydial gene products that elicit the synovial inflammatory response during both active and quiescent disease, although much more study will be required to complete the understanding of that complex process of host-pathogen interaction. Several newly developed experimental methods and approaches for study of the process will enable identification of new therapeutic targets, and possibly strategies for prevention of the disease altogether.