Technical comment on "Synovial fibroblast-neutrophil interactions promote pathogenic adaptive immunity in rheumatoid arthritis".

Reassessment of citrullinome cargo in neutrophil extracellular traps confirms the presence of citrullinated peptides.

Specimen Collection for Translational Studies in Hidradenitis Suppurativa.

Hidradenitis suppurativa (HS) is a chronic inflammatory disorder characterized by painful nodules, sinus tracts, and scars occurring predominantly in intertriginous regions. The prevalence of HS is currently 0.053-4%, with a predominance in African-American women and has been linked to low socioeconomic status. The majority of the reported literature is  retrospective, population based, epidemiologic studies. In this regard, there is a need to establish a repository of biospecimens, which represent appropriate gender and racial demographics amongst HS patients. These efforts will diminish knowledge gaps in understanding the disease pathophysiology. Hence, we sought to outline a step-by-step protocol detailing how we established our HS biobank to facilitate the formation of other HS tissue banks. Equipping researchers with carefully detailed processes for collection of HS specimens would accelerate the accumulation of well-organized human biological material. Over time, the scientific community will have access to a broad range of HS tissue biospecimens, ultimately leading to more rigorous basic and translational research. Moreover, an improved understanding of the pathophysiology is necessary for the discovery of novel therapies for this debilitating disease. We aim to provide high impact translational research methodology for cutaneous biology research and foster multidisciplinary collaboration and advancement of our understanding of cutaneous diseases.

Pathogenic immunity in systemic lupus erythematosus and atherosclerosis: common mechanisms and possible targets for intervention.

Systemic lupus erythematosus (SLE) is an autoimmune disorder that primarily affects young women and is characterized by inflammation in several organs including kidneys, skin, joints, blood and nervous system. Abnormal immune cellular and humoral responses play important roles in the development of the disease process. Impaired clearance of apoptotic material is a key factor contributing to the activation of self-reactive immune cells. The incidence of atherosclerotic cardiovascular disease (CVD) is increased up to 50-fold in patients with SLE compared to age- and gender-matched controls, and this can only partly be explained by traditional risk factors for CVD. Currently, there is no effective treatment to prevent CVD complications in SLE. Traditional preventive CVD therapies have not been found to significantly lower the incidence of CVD in SLE; therefore, there is a need for novel treatment strategies and increased understanding of the mechanisms involved in the pathogenesis of CVD complications in SLE. The pathogenic immune responses in SLE and development of atherosclerotic plaques share some characteristics, such as impaired efferocytosis and skewed T-cell activation, suggesting the possibility of identifying novel targets for intervention. As novel immune-based therapies for CVD are being developed, it is possible that some of these may be effective for the prevention of CVD and for immunomodulation in SLE. However, further understanding of the mechanisms leading to an increased prevalence of cardiovascular events in SLE is critical for the development of such therapies.

Effects of prasterone (dehydroepiandrosterone) on markers of cardiovascular risk and bone turnover in premenopausal women with systemic lupus erythematosus: a pilot study.

DHEA (dehydroepiandrosterone) is a weak androgen with proposed efficacy in the treatment of mild to moderate lupus, and possible beneficial effects on cardiovascular risk and bone mineral density. We hypothesized that treatment with 200 mg a day of Prasterone (DHEA) would improve pre-clinical measures of atherosclerosis: flow-mediated dilatation (FMD), nitroglycerin-mediated dilatation (NMD), and circulating apoptotic endothelial cells (CD 146(AnnV +)), as well markers of bone metabolism. Thirteen premenopausal female patients with Systemic Lupus Erythematosus Disease Activity Index (SLEDAI)

Lupus-prone New Zealand Black/New Zealand White F1 mice display endothelial dysfunction and abnormal phenotype and function of endothelial progenitor cells.

Patients with systemic lupus erythematosus (SLE) have an impairment in phenotype and function of endothelial progenitor cells (EPCs) which is mediated by interferon alpha (IFN-alpha). We assessed whether murine lupus models also exhibit vasculogenesis abnormalities and their potential association with endothelial dysfunction. Phenotype and function of EPCs and type I IFN gene signatures in EPC compartments were assessed in female New Zealand Black/New Zealand White F(1) (NZB/W), B6.MRL-Fas(lpr)/J (B6/lpr) and control mice. Thoracic aorta endothelial and smooth muscle function were measured in response to acetylcholine or sodium nitropruside, respectively. NZB/W mice displayed reduced numbers, increased apoptosis and impaired function of EPCs. These abnormalities correlated with significant decreases in endothelium-dependent vasomotor responses and with increased type I IFN signatures in EPC compartments. In contrast, B6/lpr mice showed improvement in endothelium-dependent and endothelial-independent responses, no abnormalities in EPC phenotype or function and downregulation of type I IFN signatures in EPC compartments. These results indicate that NZB/W mice represent a good model to study the mechanisms leading to endothelial dysfunction and abnormal vasculogenesis in lupus. These results further support the hypothesis that type I IFNs may play an important role in premature vascular damage and, potentially, atherosclerosis development in SLE.

Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma.

Like many epithelial tumors, head and neck squamous cell carcinoma (HNSCC) contains a heterogeneous population of cancer cells. We developed an immunodeficient mouse model to test the tumorigenic potential of different populations of cancer cells derived from primary, unmanipulated human HNSCC samples. We show that a minority population of CD44(+) cancer cells, which typically comprise <10% of the cells in a HNSCC tumor, but not the CD44(-) cancer cells, gave rise to new tumors in vivo. Immunohistochemistry revealed that the CD44(+) cancer cells have a primitive cellular morphology and costain with the basal cell marker Cytokeratin 5/14, whereas the CD44(-) cancer cells resemble differentiated squamous epithelium and express the differentiation marker Involucrin. The tumors that arose from purified CD44(+) cells reproduced the original tumor heterogeneity and could be serially passaged, thus demonstrating the two defining properties of stem cells: ability to self-renew and to differentiate. Furthermore, the tumorigenic CD44(+) cells differentially express the BMI1 gene, at both the RNA and protein levels. By immunohistochemical analysis, the CD44(+) cells in the tumor express high levels of nuclear BMI1, and are arrayed in characteristic tumor microdomains. BMI1 has been demonstrated to play a role in self-renewal in other stem cell types and to be involved in tumorigenesis. Taken together, these data demonstrate that cells within the CD44(+) population of human HNSCC possess the unique properties of cancer stem cells in functional assays for cancer stem cell self-renewal and differentiation and form unique histological microdomains that may aid in cancer diagnosis.

MR imaging in two cases of subacute denervation change in the muscles of facial expression.

SUMMARY: Denervation changes in muscle following damage to cranial and peripheral nerves can be observed on both CT and MR imaging studies. These findings are well described for cranial nerves (CN) V, X, XI, and XII. The CT findings of denervation atrophy due to CN VII dysfunction have been reported. We describe the MR imaging findings in two patients with perineural spread of tumor along CN VII. Both patients showed T2 prolongation and postcontrast enhancement in muscles of facial expression, suggestive of subacute denervation changes.

Decreased Ras-mitogen-activated protein kinase signaling may cause DNA hypomethylation in T lymphocytes from lupus patients.

OBJECTIVE: Previous studies have shown that inhibiting T cell DNA methylation causes a lupus-like disease by modifying gene expression. T cells from patients with lupus exhibit diminished levels of DNA methyltransferase (MTase) enzyme activity, hypomethylated DNA, and changes in gene expression similar to those exhibited by T cells treated with methylation inhibitors, suggesting that DNA hypomethylation may contribute to human lupus. Since it is known that DNA MTase levels are regulated by the ras-mitogen-activated protein kinase (MAPK) pathway, this study sought to determine whether decreased ras-MAPK signaling could account for the DNA hypomethylation in lupus T cells. METHODS: DNA MTase messenger RNA (mRNA) from lupus patients and from healthy controls was quantitated by Northern analysis, and ras-MAPK signaling was determined by immunoblotting with antibodies to the activated forms of extracellular receptor-associated kinase (ERK). Results were compared with those in T cells in which ras-MAPK signaling was inhibited with a soluble inhibitor of MAPK ERK I (MEK1). RESULTS: T cells from patients with active lupus had diminished DNA MTase mRNA levels and decreased signaling through the ras-MAPK pathway. Inhibiting signaling through the ras-MAPK pathway with the MEK1 inhibitor decreased DNA MTase mRNA and enzyme activity to the levels seen in lupus T cells, and resulted in DNA hypomethylation resembling that seen in lupus T cells. CONCLUSION: These results suggest that a decrease in signaling through the ras-MAPK pathway may be responsible for the decreased MTase activity and DNA hypomethylation in patients with lupus.

Leflunomide Aventis Pharma.

Hoechst Marion Roussel (HMR; now Aventis Pharma) launched leflunomide (HWA-486), an immunomodulator and a disease-modifying antirheumatic drug (DMARD), for the treatment of rheumatoid arthritis (RA) in the US in late 1998 [310118]. By August 2000, the compound had been launched extensively across all of Latin America and in all major European countries [380046]. The compound is also under preclinical investigationfor the prevention of transplant rejection [279727], [304402]. In 1998, HMR filed for approvalfor RA in Europe [279727]. In September 1998, the FDA approved leflunomide for the treatment of active RA in adults and it was launched shortly thereafter [298204], [299258], [310118]. In September 1999, the EU Commission accepted the view of the Committee for Proprietary Medicinal Products, published in May 1999 [326040], [337534], and gave approval for the use of leflunomide in RA in adults [339128]. Lehman Brothers has reported that EU launch was delayed by rare side effects including pancytopenia [354434]. In August 1998, the Arthritis Advisory Committee unanimously recommended that leflunomide be contraindicatedfor pregnancy, and that a pregnancy registry should be established to monitor possible teratogenic effects of the drug [296187]. Kyorin had a licence to develop leflunomide in Japan. Product approval was scheduled for 1998 [159079], but no development has been reported since 1994. Preclinical studies in an animal model of experimental allergic encephalomyelitis (EAE) have shown leflunomide to be a powerful immunosuppressant which may have potential in diseases such as multiple sclerosis [187881]. Leflunomide is rapidly processed in vivo to its active metabolite, A-771726 (RS-61980) [202941], [253615]. In 1996, leflunomide was designated as one of HMR's nine top-priority products, serving an unmet medical need and addressing a potential market in excess of US $500 million per year [221118].

Impaired translational response and increased protein kinase PKR expression in T cells from lupus patients.

Activation of peripheral blood T cells results in a rapid and substantial rise in translation rates and proliferation, but proliferation in response to mitogen stimulation is impaired in systemic lupus erythematosus (SLE). We have investigated translation rates and initiation factor activities in T cells from SLE patients in response to activating signals. Activation by PMA plus ionomycin strongly increased protein synthesis in control T cells but not in T cells from SLE patients. The rate of protein synthesis is known to be strongly dependent on the activity of two eukaryotic translation initiation factors, eIF4E and eIF2alpha. We show that following stimulation, eIF4E expression and phosphorylation increased equivalently in control and SLE T cells. Expression of eIF4E interacting proteins - eIF4G, an inducer, and 4E-BP1 and 4E-BP2, two specific repressors of eIF4E function - and the phosphorylation level of 4E-BP1, were all identical in control and SLE T cells. In contrast, the protein kinase PKR, which is responsible for the phosphorylation and consequent inhibition of eIF2alpha activity, was specifically overexpressed in activated SLE T cells, correlating with an increase in eIF2alpha phosphorylation. Therefore, high expression of PKR and subsequent eIF2alpha phosphorylation is likely responsible, at least in part, for impaired translational and proliferative responses to mitogens in T cells from SLE patients.

Childhood emotional trauma and chronic posttraumatic stress disorder in adult outpatients with treatment-resistant depression.

The intent of this study was to test the hypothesis that patients with treatment-resistant depression are more likely than treatment responsive patients to suffer from sequelae of childhood trauma that may perpetuate depression despite adequate medication treatment. Twenty participants with treatment-resistant depression and 20 participants with treatment-responsive depression were administered a structured interview and a battery of psychological tests to assess levels of current depression, confirm diagnosis, and quantify childhood trauma and presence of dissociative phenomena. Tests used include the Beck Depression Inventory, the Mini International Neuropsychiatric Interview, the Minnesota Multiphasic Personality Inventory-2, the Childhood Trauma Questionnaire, and the Trauma Symptom Inventory. Compared with treatment responders, the treatment-resistant participants were significantly more depressed, had significantly more comorbid anxiety disorders, reported significantly greater levels of childhood emotional abuse, and experienced current-day sequelae of childhood emotional abuse. The hypothesis was partially supported by these results. This study suggests that reported history of childhood emotional abuse and sequelae of that abuse may be associated with treatment resistance in depressed outpatients.

Thrombosis in patients with connective tissue diseases treated with specific cyclooxygenase 2 inhibitors. A report of four cases.

Specific inhibitors of cyclooxygenase 2 (COX-2) have been approved for the treatment of osteoarthritis and rheumatoid arthritis. Unlike nonsteroidal anti-inflammatory drugs, specific COX-2 inhibitors do not inhibit platelet activation. However, these agents significantly reduce systemic production of prostacyclin. As a result, theoretical concerns have been raised that specific COX-2 inhibitors could shift the hemostatic balance toward a prothrombotic state. Patients with connective tissue diseases (CTD), who may be predisposed to vasculopathy and thrombosis, often have arthritis or pain syndromes requiring treatment with antiinflammatory agents. Herein we describe 4 patients with CTD who developed ischemic complications after receiving celecoxib. All patients had a history of Raynaud's phenomenon, as well as elevated anticardiolipin antibodies, lupus anticoagulant, or a history compatible with antiphospholipid syndrome. It was possible to measure a urinary metabolite of thromboxane A2 in 2 of the patients as an indicator of in vivo platelet activation, and this was markedly elevated in both. In addition, the patients had evidence of ongoing inflammation as indicated by elevated erythrocyte sedimentation rate, hypocomplementemia, and/or elevated levels of anti-DNA antibodies. The findings in these 4 patients suggest that COX-2 inhibitor-treated patients with diseases that predispose to thrombosis should be monitored carefully for this complication.

Recurrence of clival chordoma along the surgical pathway.

Chordomas are locally aggressive malignant tumors of notochordal origin whose metastatic potential is increasingly recognized. Surgical pathway recurrence has been noted only rarely in the literature. We present three patients with clival chordomas whose sole or initial recurrence was along the pathway of prior surgical access. A characteristic mass found along the pathway of prior surgical access for resection of a chordoma should suggest recurrent chordoma.

Lymph node metastasis in maxillary sinus carcinoma.

PURPOSE: To evaluate the incidence and prognostic significance of lymph node metastasis in maxillary sinus carcinoma. METHODS AND MATERIALS: We reviewed the records of 97 patients treated for maxillary sinus carcinoma with radiotherapy at Stanford University and at the University of California, San Francisco between 1959 and 1996. Fifty-eight patients had squamous cell carcinoma (SCC), 4 had adenocarcinoma (ADE), 16 had undifferentiated carcinoma (UC), and 19 had adenoid cystic carcinoma (AC). Eight patients had T2, 36 had T3, and 53 had T4 tumors according to the 1997 AJCC staging system. Eleven patients had nodal involvement at diagnosis: 9 with SCC, 1 with UC, and 1 with AC. The most common sites of nodal involvement were ipsilateral level 1 and 2 lymph nodes. Thirty-six patients were treated with definitive radiotherapy alone, and 61 received a combination of surgical and radiation treatment. Thirty-six patients had neck irradiation, 25 of whom received elective neck irradiation (ENI) for N0 necks. The median follow-up for alive patients was 78 months. RESULTS: The median survival for all patients was 22 months (range: 2.4-356 months). The 5- and 10-year actuarial survivals were 34% and 31%, respectively. Ten patients relapsed in the neck, with a 5-year actuarial risk of nodal relapse of 12%. The 5-year risk of neck relapse was 14% for SCC, 25% for ADE, and 7% for both UC and ACC. The overall risk of nodal involvement at either diagnosis or on follow-up was 28% for SCC, 25% for ADE, 12% for UC, and 10% for AC. All patients with nodal involvement had T3-4, and none had T2 tumors. ENI effectively prevented nodal relapse in patients with SCC and N0 neck; the 5-year actuarial risk of nodal relapse was 20% for patients without ENI and 0% for those with elective neck therapy. There was no correlation between neck relapse and primary tumor control or tumor extension into areas containing a rich lymphatic network. The most common sites of nodal relapse were in the ipsilateral level 1-2 nodal regions (11/13). Patients with nodal relapse had a significantly higher risk of distant metastasis on both univariate (p = 0.02) and multivariate analysis (hazard ratio = 4.5, p = 0.006). The 5-year actuarial risk of distant relapse was 29% for patients with neck control versus 81% for patients with neck failure. There was also a trend for decreased survival with nodal relapse. The 5-year actuarial survival was 37% for patients with neck control and 0% for patients with neck relapse. CONCLUSION: The overall incidence of lymph node involvement at diagnosis in patients with maxillary sinus carcinoma was 9%. Following treatment, the 5-year risk of nodal relapse was 12%. SCC histology was associated with a high incidence of initial nodal involvement and nodal relapse. None of the patients presenting with SCC histology and N0 necks had nodal relapse after elective neck irradiation. Patients who had nodal relapse had a higher risk of distant metastasis and poorer survival. Therefore, our present policy is to consider elective neck irradiation in patients with T3-4 SCC of the maxillary sinus.

TRAIL (Apo2 ligand) and TWEAK (Apo3 ligand) mediate CD4+ T cell killing of antigen-presenting macrophages.

The human marrow produces approximately 1010 monocytes daily, and this production must be balanced by a similar rate of destruction. Monocytes/macrophages can undergo apoptosis after activating CD4+ T cells, suggesting one mechanism that may contribute to macrophage homeostasis. Previous reports indicate that Fas-Fas ligand interactions are the principle molecules mediating this response. However, D10, an Iak-restricted cloned Th2 line, will similarly induce apoptosis in Ag-presenting macrophages, and D10 cells lack Fas ligand. To confirm that D10 cells kill macrophages through Fas-independent pathways, D10 cells were shown to kill MRL lpr/lpr (Iak) macrophages in an Ag-dependent fashion, indicating additional mechanisms. Recent reports demonstrate that TNF-related apoptosis-inducing ligand (TRAIL), interacting with Apo2, and TNF-like weak inducer of apoptosis (TWEAK), interacting with Apo3, will induce apoptosis in some cells. Using Abs to TRAIL and an Apo3-IgG Fc fusion protein, we demonstrated that D10 cells express both TRAIL and TWEAK. The Apo3 fusion protein, but not human IgG, inhibited D10-induced macrophage apoptosis, as did anti-TRAIL. Further studies demonstrated that AE7, a cloned Th1 line, and splenic T cells express TWEAK, TRAIL, and Fas ligand, and inhibiting these molecules also inhibited macrophage killing. These results indicate that D10 cells induce macrophage apoptosis through TRAIL- and TWEAK-dependent pathways. Because normal T cells also express these molecules, these results support the concept that T cells have multiple pathways by which to induce macrophage apoptosis. These pathways may be important in immune processes such as macrophage homeostasis as well as in down-regulation of immune responses and elimination of macrophages infected with intracellular organisms.

LFA-1 overexpression and T cell autoreactivity: mechanisms.

Overexpressing LFA-1 (CD11a/CD18) on antigen specific CD4+ T cells makes the cells proliferate to normally subthreshold stimuli, including self-Ia molecules without specific antigen. The mechanisms by which this occurs are unknown, but potentially include transmission of an increased costimulatory signal, overstabilization of normally low affinity TCR-Ia interactions, or both. A role for increased costimulatory signaling was tested by culturing control and CD18-transfected antigen-specific T cells clones with anti-CD3 and anti-CD11a. Minimal calcium fluxes were detected, but increased protein tyrosine phosphorylation was observed in the transfectants. However, the proliferative response to graded amounts of these antibodies were identical in the transfectants and controls, suggesting that increased signaling alone was insufficient to cause the increased responsiveness. To test for overstabilization, transfected and control clones were cultured with syngeneic Mø with or without antigen. The transfected but not control cells downregulated TCR expression in response to Mø alone, thus demonstrating successful TCR signaling to a low affinity interaction. These results indicate that LFA-1 overexpression permits TCR signal transmission to a normally subthreshold stimulus presented by Mø, consistent with overstabilization. LFA-1 overexpression also causes increased tyrosine phosphorylation, but this alone is not sufficient to cause a proliferative response to low level stimuli.

DNA methylation in the regulation of T cell LFA-1 expression.

Inducing T cell LFA-1 overexpression by transfection, or by treatment with DNA methylation inhibitors including 5-azacytidine, procainamide and hydralazine, causes MHC-specific T cell autoreactivity in vitro and autoimmunity in vivo. How DNA methylation inhibitors increase LFA-1 expression is unknown. In this report we identify a mechanism by which DNA methylation affects LFA-1 expression. Nuclear run-on assays demonstrated that inhibiting DNA methylation increased transcription of CD11a but not CD18 or beta-actin mRNA. CD11a mRNA stability was not affected. Transfection of hypomethylated cells with reporter constructs containing the CD11a promoter showed no role for overexpression of transcription factors. However, the CD11a promoter demethylated following treatment with DNA methyltransferase inhibitors, and in vitro methylation of the construct suppressed its expression. Together, these results indicate that DNA methylation inhibitors can cause LFA-1 overexpression directly by demethylating the CD11a promoter. This mechanism could contribute to T cell autoreactivity, and potentially to autoimmunity.

Diffuse swelling of one finger in a patient with metastatic breast cancer.

Tumor metastases distal to the elbow, and specifically in the hand, are extremely rare. The diagnosis can be difficult, because these lesions can resemble those of rheumatoid arthritis, other inflammatory arthritis, and osteomyelitis. Therefore, rheumatologists should consider metastasis to the limbs in the differential diagnosis of individuals who present with joint or bone manifestations and history of cancer, even if remote. We report a woman with breast cancer and metastatic disease predominantly affecting one finger.

Systemic toxicity following administration of sirolimus (formerly rapamycin) for psoriasis: association of capillary leak syndrome with apoptosis of lesional lymphocytes.

BACKGROUND: Sirolimus (formerly rapamycin) is an immunosuppressive agent that interferes with T-cell activation. After 2 individuals with psoriasis developed a capillary leak syndrome following treatment with oral sirolimus lesional skin cells and activated peripheral blood cells were analyzed for induction of apoptosis. OBSERVATIONS: A keratome skin specimen from 1 patient with sirolimus-induced capillary leak syndrome had a 2.3-fold increase in percentage of apoptotic cells (to 48%) compared with an unaffected sirolimus-treated patient with psoriasis (21%). Activated peripheral blood T cells from patients with psoriasis tended to exhibit greater spontaneous or dexamethasone-induced apoptosis than did normal T cells, particularly in the presence of sirolimus. CONCLUSIONS: Severe adverse effects of sirolimus include fever, anemia, and capillary leak syndrome. These symptoms may be the result of drug-induced apoptosis of lesional leukocytes, especially activated T lymphocytes, and possibly release of inflammatory mediators. Because patients with severe psoriasis may develop capillary leak from various systemic therapies, clinical monitoring is advisable for patients with inflammatory diseases who are treated with immune modulators.

Metastatic head and neck cancer: role and usefulness of FDG PET in locating occult primary tumors.

PURPOSE: To assess the usefulness of 2-[fluorine-18]fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET) of the head and neck in locating occult primary lesions in patients with metastatic cervical adenopathy. MATERIALS AND METHODS: Seventeen patients with metastatic cervical adenopathy of unknown primary origin were referred for FDG PET of the head and neck. All patients had undergone correlative anatomic imaging within 1 month of FDG PET. Surgical, clinical, and histopathologic findings were used to assess the performance of FDG PET. RESULTS: Increased apical lung uptake at FDG PET led to a biopsy-proved diagnosis of primary lung cancer in two patients. Of the remaining 15 patients, 10 had a focus of increased activity; directed biopsy of these sites led to confirmation of a primary carcinoma in seven patients. Correlative anatomic imaging failed to demonstrate the primary sites of disease in two of these seven patients. None of the five patients with negative FDG PET studies have manifested evidence of a primary site of disease during follow-up of 8-42 months (mean, 29 months). CONCLUSION: FDG PET allows effective localization of the unknown primary site of origin in metastatic head and neck cancer and can contribute substantially to patient care.