Richard C. Chou

Lipid-Cytokine-Chemokine Cascade Drives Neutrophil Recruitment in a Murine Model of Inflammatory Arthritis

A large and diverse array of chemoattractants control leukocyte trafficking, but how these apparently redundant signals collaborate in vivo is still largely unknown. We previously demonstrated an absolute requirement for the lipid chemoattractant leukotriene B(4) (LTB(4)) and its receptor BLT1 for neutrophil recruitment into the joint in autoantibody-induced arthritis. We now demonstrate that BLT1 is required for neutrophils to deliver IL-1 into the joint to initiate arthritis. IL-1-expressing neutrophils amplify arthritis through the production of neutrophil-active chemokines from synovial tissue cells. CCR1 and CXCR2, two neutrophil chemokine receptors, operate nonredundantly to sequentially control the later phase of neutrophil recruitment into the joint and mediate all neutrophil chemokine activity in the model. Thus, we have uncovered a complex sequential relationship involving unique contributions from the lipid mediator LTB(4), the cytokine IL-1, and CCR1 and CXCR2 chemokine ligands that are all absolutely required for effective neutrophil recruitment into the joint.

Synchronization of Mammalian Cell Cultures by Serum Deprivation

Mammalian cells are amenable to study the regulation of cell cycle progression in vitro by shifting them into the same phase of the cycle. Procedures to arrest cultured cells in specific phases of the cell cycle may be termed in vitro synchronization. The procedure described here was developed for the study of primary astrocytes and a glioma cell line, but is applicable to other mammalian cells. Its application allows astrocytes to reenter the cell cycle from a state of quiescence (G(0)), and then, under carefully defined experimental conditions, to move together into subsequent phases such as the G(1) and S phases. A number of methods have been established to synchronize mammalian cell cultures, which include physical separation by centrifugal elutriation and mitotic shake off or chemically induced cell cycle arrest. Yet, there are intrinsic limitations associated with these methods. In the present protocol, we describe a simple, reliable, and reversible procedure to synchronize astrocyte and glioma cultures from newborn rat brain by serum deprivation. The procedure is similar, and generally applicable, to other mammalian cells. This protocol consists essentially of two parts: (1) proliferation of astrocytes under optimal conditions in vitro until reaching desired confluence; and (2) synchronization of cultures by serum downshift and arrested in the G(0) phase of the cell cycle. This procedure has been extended to the examination of cell cycle control in astroglioma cells and astrocytes from injured adult brain. It has also been employed in precursor cloning studies in developmental biology, suggesting wide applicability.

Therapeutic monoclonal antibodies and derivatives: Historical perspectives and future directions.

Biologics, both monoclonal antibodies (mAbs) and fusion proteins, have revolutionized the practice of medicine. This year marks the 30th anniversary of the Food and Drug Administration approval of the first mAb for human use. In this review, we examine the biotechnological breakthroughs that spurred the explosive development of the biopharmaceutical mAb industry, as well as how critical lessons learned about human immunology informed the development of improved biologics. We also discuss the most common mechanisms of action of currently approved biologics and the indications for which they have been approved to date.

Successful treatment of rheumatoid meningitis with cyclophosphamide but not infliximab

Rheumatoid meningitis is a rare but lethal disorder that occurs in an elderly population with longstanding rheumatoid arthritis (RA).1 Although the majority of patients experience neurological symptoms, up to 26% of the patients were asymptomatic in a case series study as identified by necropsy.1 There is poor correlation between the severity of synovitis and neurological symptoms, which therefore imposes a challenge in the diagnosis of this condition.1 To date there are no established treatment regimens for rheumatoid meningitis, although most patients receive immunosuppressive agents. Although the anti-tumour necrosis factor (TNF) agents have been proved to provide significant relief for the articular manifestations of RA, their effectiveness for rheumatoid meningitis has not been reported.2 A 58 year old woman with previous diagnoses of fibromyalgia and osteoarthritis was referred to the rheumatology clinic of the Massachusetts General Hospital (MGH) with worsening polyarthritis of both hands, wrists, knees, and ankles while receiving daily rofecoxib, 25 mg. Before the visit she had undergone extensive investigation for a 6 …

A 36-year-old woman with recurrent high-grade fevers, hypotension, and hypertriglyceridemia.

History of the present illness The patient had been admitted 6 months earlier to an outside hospital with a high-grade fever, an erythematous rash on her lower extremities, and pain in her legs. She had a history of bipolar disorder and schizoaffective disorder, and had been treated with the antipsychotic medications ziprasidone and quetiapine fumarate. An initial evaluation for infections was negative, and broadspectrum antibiotics failed to relieve the fever. The patient’s fevers were attributed to the neuroleptic malignant syndrome (Table 1). Her fevers continued unabated to temperatures as high as 103°F, despite discontinuation of her antipsychotic medications and treatment with bromocriptine. She was transferred to another hospital for further evaluation and management. At the transfer hospital, the patient exhibited a transient increase in her creatine kinase level to 1,389 units/liter (normal value 240). Assays for antinuclear antibodies, antibodies to double-stranded DNA, and extractable nuclear antigens (Ro, La, Sm, and RNP) were negative, as were anti–Jo-1 antibodies. A magnetic resonance image of the lower leg showed muscle edema with localized infarction. A muscle biopsy showed perivascular inflammatory infiltrates consistent with an inflammatory myopathy. Prednisone 60 mg/day was started and azathioprine was considered, but the patient’s localized rash and leg pain resolved spontaneously before treatment with azathioprine was started. The patient was prescribed a tapering course of prednisone for presumed dermatomyositis, and did not experience recurrent episodes of fever, rash, or leg pain while receiving glucocorticoids (Table 1). No photographic record of the patient’s original rash exists. Within one week of discharge, the patient developed recurrent fevers and was readmitted to a third hospital. Her dose of prednisone at that time was unknown. She was later found to have a urinary tract infection caused by Escherichia coli and was treated with trimethoprim/sulfamethoxazole. Despite antibiotic treatment, the patient continued to have intermittent high-grade fevers and episodes of hypotension, with blood pressure as low as 70/40 mm Hg (Table 1). She was transferred to a fourth hospital. Upon transfer, the patient had a temperature of 104°F, sinus tachycardia (140–160 beats/minute), hypotension, and a hematocrit of 20%. There was no rash despite the development of intermittent high-grade fever. Multiple cultures were negative for infection. The patient underwent a bone marrow biopsy that showed a hypercellular marrow, and a peripheral smear showed some dysplastic myeloid forms and blasts. These were interpreted as being consistent with reactive changes associated with anemia of chronic disease. A second muscle biopsy finding showed mild perivascular inflammatory infiltrates in the endomysium, consistent with an inflammatory myopathy. However, further analysis by electron microscopy and immunohistochemical staining demonstrated no definitive evidence of such a condition. Antibodies against the membrane attack complex of complement, the major histocompatibility complex, CD20, CD3, laminin , Brown-Brenn stain, and NADH reductase were negative. The final interpretation of the muscle biopsy finding was nonspecific and possibly the result of trauma or ischemia at needle entry sites. The patient continued receiving prednisone without a Richard C. Chou, MD, PhD (current address: DartmouthHitchcock Medical Center, Lebanon, New Hampshire), Judith A. Ferry, MD: Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts; Charles A. Dinarello, MD: University of Colorado School of Medicine in Denver, Aurora; Paola Dal Cin, PhD: Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts. Address correspondence to Richard C. Chou, MD, PhD, Section of Rheumatology, Department of Medicine, Dartmouth-Hitchcock Medical Center, 1 Medical Center Drive, Lebanon, NH 03756. E-mail: Richard.C.Chou@hitchcock. org. Submitted for publication January 29, 2009; accepted in revised form September 14, 2009. Arthritis Care & Research Vol. 62, No. 1, January 15, 2010, pp 128–136 DOI 10.1002/acr.20024

Ehlers-Danlos syndrome hypermobility type is associated with rheumatic diseases

We retrospectively analyzed electronic medical records of patients with Ehlers-Danlos Syndrome hypermobility type (HEDS), including demographic information, workup, rheumatological diagnoses in order to determine its association with rheumatological conditions. HEDS Patients were stratified according to level of workup received (no additional work (physical exam only) = NWU, limited workup = LWU, comprehensive workup = CWU)). HEDS patients were predominantly female (21:4, F:M). The percentage of patients with at least one rheumatological condition was significantly correlated with level of workup (NWU, 9.2%; LWU, 33.3%, CWU, 67.1%; p-value < 0.0001). The HLA-B27 antigen was more prevalent (p-value < 2.2 × 10–8) in the CWU HEDS patients (23.9%) than in the general population of the United States (6.1%). HEDS with CWU were associated with more rheumatological conditions (i.e. psoriasis, ankylosing spondylitis, rheumatoid arthritis, fibromyalgia) than those with NWU or LWU. In conclusion, HEDS is associated with complicated rheumatological conditions, which are uncovered by comprehensive workup. These conditions require different clinical management strategies than HEDS, and left untreated could contribute to the pain or even physical disability (i.e. joint erosions) in HEDS patients. While the mechanisms underlying these associations are unknown, it is important that all HEDS patients receive adequate workup to ensure a complete clinical understanding for the best care strategy possible.

Non-aggregated Aβ25-35 Upregulates Primary Astrocyte Proliferation In Vitro

Amyloid beta (Aβ) is a peptide cleaved from amyloid precursor protein that contributes to the formation of senile plaques in Alzheimer’s disease (AD). The relationship between Aβ and astrocyte proliferation in AD remains controversial. Despite pathological findings of increased astrocytic mitosis in AD brains, in vitro studies show an inhibitory effect of Aβ on astrocyte proliferation. In this study, we determined the effect of an active fragment of Aβ (Aβ25-35) on the cell cycle progression of primary rat astrocytes. We found that Aβ25-35 (0.3–1.0 μg/ml) enhanced astrocyte proliferation in vitro in a time- and concentration-dependent manner. Increased DNA synthesis by Aβ25-35 was observed during the S phase of the astrocyte cell cycle, as indicated by proliferation kinetics and bromodeoxyuridine immunocytochemical staining. Aggregation of Aβ25-35 abolished the upregulatory effect of Aβ on astrocyte proliferation. Further examination indicated that Aβ25-35 affected astrocyte proliferation during early or mid-G1 phase but had no effect on DNA synthesis at the peak of S phase. These results provide insight into the relationship between Aβ25-35 and astrocyte cell cycling in AD.

Differential and kinetic effects of cell cycle inhibitors on neoplastic and primary astrocytes

ABSTRACT Alterations in cell cycle regulation underlie the unrestricted growth of neoplastic astrocytes. Chemotherapeutic interventions of gliomas have poor prognostic outcomes due to drug resistance and drug toxicity. Here, we examined the in vitro growth kinetics of C6 glioma (C6G) cells and primary astrocytes and their responses to 2 phase-specific inhibitors, lovastatin and hydroxyurea. C6G cells demonstrated a shorter G1 phase and an earlier peak of DNA synthesis in S phase than primary astrocytes. As C6G cells and primary astrocytes re-entered the cell cycle in the presence of lovastatin or hydroxyurea, they exhibited different sensitivities to the inhibitory effects of these agents, as measured by [3H]-thymidine incorporation. Compared to primary astrocytes, C6G cells were more sensitive to lovastatin, but less sensitive to hydroxyurea. Studies using 2 different paradigms of exposure uncovered dramatic differences in the kinetics of DNA synthesis inhibition by these 2 agents in C6G cells and primary astrocytes. One notable difference was the ability of C6G cells to more easily recover from the inhibitory effects of hydroxyurea following short exposure. Our results provide insight into C6 glioma drug resistance as well as the inhibitory effects of these 2 phase-specific inhibitors and their chemotherapeutic potential.

A 44-year-old woman with right ankle pain

History of the present illness Eleven months prior to her rheumatology clinic visit, the patient developed right ankle pain unrelated to weight bearing. Within a month, the right ankle pain had spread to the other ankle. The pain was associated with ankle swelling and the sensation of tingling in her feet. Two months after these initial symptoms, pain and stiffness developed in her hands and wrists. She denied swelling of these areas. The pain extended subsequently to her shoulders and elbows. The patient was evaluated at a local urgent care center. She experienced pain in multiple upper and lower extremity joints, but there was no synovitis, soft tissue swelling, or signs of joint effusion. Despite additional symptoms of tingling and pain in her ankles and feet, the strength in her extremities was normal and the sensory examination was intact to light touch. A complete blood cell count and serum chemistry profile were both normal. The patient was sent home on naproxen and instructed to follow up with her primary care doctor. The primary care doctor evaluated the patient 2 weeks later and reviewed additional serologies ordered in the urgent care center. The patient’s serum was positive for rheumatoid factor (RF; titer 258 IU, normal value 30). Assays for antibodies to cyclic citrullinated peptides (antiCCPs) and antinuclear antibodies were negative. The patient’s erythrocyte sedimentation rate (ESR) was 96 mm/ hour (normal value 20) and her serum C-reactive protein (CRP) concentration was 50.6 mg/liter (normal value 8.0). The primary care provider diagnosed rheumatoid arthritis (RA) and prescribed prednisone 30 mg/day, ibuprofen 800 mg 3 times daily, oxycodone 2.5 mg twice daily, and 2 tablets of acetaminophen with codeine at bedtime as needed. The patient felt somewhat better on this regimen, but over the next 8 months she was unable to taper her prednisone dosage below 30 mg/day because of worsening pain in her extremities and sensations of numbness and tingling in her ankles and feet. She also continued her ibuprofen, oxycodone, and acetaminophen with codeine for pain control. After months of this regimen, the patient developed a purpuric rash over her right lower leg. She was referred to the rheumatology clinic. The patient’s past medical history was remarkable for vitiligo, which she had had for years. The vitiligo had involved large areas over her distal arms, hands, legs, feet, and face. The family history was negative for autoimmune and inflammatory conditions. She did not smoke or drink. She had 3 children, was divorced, and worked as a housekeeper. On physical examination, the patient had a blood pressure of 143/84 mm Hg. Her temperature was 98.0°C, her respiratory rate was 16/minute, and her pulse was 66 beats/minute. There were no findings on examination of the head, eyes, ears, nose, mouth, neck, heart, lungs, abdomen, or back. However, examination of her ankles revealed severe tenderness of the skin of both ankles. There was no synovitis, joint effusion, or soft tissue swelling. Her skin examination showed a prominent livedo racemosa rash over her lower extremities (Figure 1A), a palpable purpuric lesion on the right lower leg (Figure 1B), and diffuse hypopigmented areas secondary to vitiligo (Figure 1C). Some of the larger purpuric lesions had areas of central necrosis and ulceration. Her neurologic examination revealed numbness in the lower extremities distal to the ankles, corresponding to the symptoms of pain and tingling. The strength of all of the muscle groups was 5/5 bilaterally, with no indication of either a foot or wrist drop. The deep tendon reflexes were diminished symmetrically at the ankles. The patient’s full serologic evaluation and other laboratory test results are shown in Table 1.