James N. Woody

Therapeutic efficacy of multiple intravenous infusions of anti-tumor necrosis factor alpha monoclonal antibody combined with low-dose weekly methotrexate in rheumatoid arthritis

OBJECTIVE To evaluate the efficacy, pharmacokinetics, immunogenicity, and safety of multiple infusions of a chimeric monoclonal anti-tumor necrosis factor alpha antibody (cA2) (infliximab; Remicade, Centocor, Malvern, PA) given alone or in combination with low-dose methotrexate (MTX) in rheumatoid arthritis (RA) patients. METHODS In a 26-week, double-blind, placebo-controlled, multicenter trial, 101 patients with active RA exhibiting an incomplete response or flare of disease activity while receiving low-dose MTX were randomized to 1 of 7 groups of 14-15 patients each. The patients received either intravenous cA2 at 1, 3, or 10 mg/kg, with or without MTX 7.5 mg/week, or intravenous placebo plus MTX 7.5 mg/week at weeks 0, 2, 6, 10, and 14 and were followed up through week 26. RESULTS Approximately 60% of patients receiving cA2 at 3 or 10 mg/kg with or without MTX achieved the 20% Paulus criteria for response to treatment, for a median duration of 10.4 to >18.1 weeks (P < 0.001 versus placebo). Patients receiving cA2 at 1 mg/kg without MTX became unresponsive to repeated infusions of cA2 (median duration 2.6 weeks; P=0.126 versus placebo). However, coadministration of cA2 at 1 mg/kg with MTX appeared to be synergistic, prolonging the duration of the 20% response in >60% of patients to a median of 16.5 weeks (P < 0.001 versus placebo; P=0.006 versus no MTX) and the 50% response to 12.2 weeks (P < 0.001 versus placebo; P=0.002 versus no MTX). Patients receiving placebo infusions plus suboptimal low-dose MTX continued to have active disease, with a Paulus response lasting a median of 0 weeks. A 70-90% reduction in the swollen joint count, tender joint count, and C-reactive protein level was maintained for the entire 26 weeks in patients receiving 10 mg/kg of cA2 with MTX. In general, treatment was well tolerated and stable blood levels of cA2 were achieved in all groups, except for the group receiving 1 mg/kg of cA2 alone, at which dosage antibodies to cA2 were observed in approximately 50% of the patients. CONCLUSION Multiple infusions of cA2 were effective and well tolerated, with the best results occurring at 3 and 10 mg/kg either alone or in combination with MTX in approximately 60% of patients with active RA despite therapy with low-dose MTX. When cA2 at 1 mg/kg was given with low-dose MTX, synergy was observed. The results of the trial provide a strategy for further evaluation of the efficacy and safety of longer-term treatment with cA2.

Human T Cell Clones

T lymphocyte clones (TLCs) specific for Chlamydia trachomatis were obtained by the limiting dilution technique of in vitro activated T cells from two different donors. Most of the TLCs obtained were only able to recognize antigen together with restriction elements on DR molecules, expressed in the antigen-presenting cells (APC). A few TLCs were restricted by elements on DRw53(MT3) molecules, and one TLC by DPw4(SB4) molecules. A close relationship was found between the restriction epitopes and those that activate allogeneic T cells in mixed lymphocyte culture (MLC) tests. The results of inhibition experiments using monoclonal antibodies (Mabs) against different HLA molecules correlated closely with the restriction specificities of the TLCs. HLA Class II Restriction Elements Chlamydia-specific T lymphocyte clones (TLCs) were generated in vitro using the limiting dilution technique, as previously described l ,2. Serological HLA-A,-B,-C, and -DR typing was performed using techniques and reagents also previously described3 . Some of the cell donors were HLA-D typed with a panel of homozygous typing cells (HTCs) expressing the specificities Dw4, DwlO, Dwl3, Dwl4, Dwl5 or KT24.

Harvest of human bone marrow directly from bone

Harvest of human bone marrow directly from freshly resected bone provides purer preparations of marrow than can be obtained by the conventional technique of multiple aspirations from the iliac crests. In particular, directly harvested marrow is much less heavily contaminated with peripheral blood lymphocytes, a known source of mature T cells. Because of the possible relevance of these contaminating T cells for cadaveric bone marrow transplants, the best source of human marrow harvested directly from bone has been studied. Human bone marrow was harvested from 46 surgical specimens and 9 cadaveric tissue donors. Vertebral bodies provided the best source of bone marrow with average yields of 3.1 +/- 1.6 X 10(9) cells per vertebra. When entire ilia were removed and processed for marrow, an average of 1.6 +/- 1.0 X 10(9) cells was obtained. Surgically resected ribs yielded lower amounts of marrow with a mean cell number of 3.2 +/- 2.6 X 10(8) per rib. Isolation of bone marrow mononuclear cells from these preparations by density gradient centrifugation resulted in a loss of 45% of the starting cells. Human bone marrow was found to contain 5-6% T cells before gradient separation and these cells were immunologically competent as measured in vitro by responses to mitogens and alloantigens. This technique may be useful in obtaining human bone marrow for both immunologic studies and allogeneic transplantation.

In vitro binding studies of anti-human lymphocyte globulin: analysis of samples tested for immunosuppression in the primate skin allograft system.

Abstract We measured the rates and the quantity of antibody which binds to cultured human lymphoblasts, thymus and, in some cases, Hela cells in 28 anti-human and 2 anti-baboon lymphocyte sera and in 9 normal sera submitted to the National Naval Medical Center for immunosuppressive testing in primates. The quantity of 125 -J-labeled ALG which binds to 1 × 10 6 lymphoblasts during 60 min incubation at 37°C correlates well ( r = 0.648) with the degree of immunosuppression produced by these same ALGs in rhesus monkeys. Twenty-one of the 22 immunosuppressive sera bound ≥6 × 10 −13 moles IgG per 10 6 lymphoblasts under conditions of the test whereas none of the 9 normal sera and only 3 of the 8 non-immunosuppressive sera bound this amount. Further studies of 2 of the 3 non-immunosuppressive ALGs which contained substantial quantities of lymphoblast binding antibodies revealed that one totally lacked antibodies able to bind to human thymus cells. The other contained thymus-reactive anti-bodies but, in comparison with two ALGs known to be immunosuppressive, it contained much less thymus cell-specific antibody as well as much less antibody cross-reactive with other human cells. All unabsorbed ALGs tested, regardless of immunosuppressive ability, contained more antibodies reactive with Hela cells ot lymphoblasts than with thymus cells. Thus, the chief feature which appears to distinguish highly immunosuppressive anti-human ALGs from those with only modest or no ability to prolong graft survival in rhesus monkeys is their content of relatively large quantities of avid antibodies reactive with human thymus cells. These studies demonstrate the ability of this in vitro test not only to rapidly identify ALGs which are likely to be immunosuppressive, but more importantly, to measure directly the relative avidity, specificity, and quantity of their antibodies reactive with lymphoid and non-lymphoid target cells.