rHuTNF Alpha Research Articles

Treatment of recurrent sarcoma of the extremities by isolated limb perfusion using tumor necrosis factor alpha and melphalan.

24-60% of patients with soft tissue sarcoma shows local recurrences after treatment of the primary tumor. The event is associated with a high incidence of macroscopic or microscopic metastases and a poor survival. Our goal is to preserve a patient's functional limb by treating such cases with isolated limb perfusion (ILP) with recombinant human tumor necrosis factor alpha (rHu TNF-alpha) and melphalan, which have demonstrated a potent antitumor activity in vivo and in vitro studies. During the period November 1991 to November 1995, 10 patients with unresectable recurrent soft tissue sarcoma of the limb were treated by ILP at intermediate hyperthermia (40-40.5 degrees C) with rHu TNF-alpha and melphalan. Two patients also received recombinant interferon gamma (rIFN-gamma) before and during ILP. We used a range of 2-4 mg for rHu TNF-alpha and 50-100 mg of melphalan. rIFN-gamma was administered on days -2 and -1 (15 x 10(6) IU) subcutaneously and the same dose was injected in the arterial line during ILP. No perioperative surgical complication was observed. Local toxicity was moderate (grade I or II); general toxicity was observed in 6 patients (2 grade I and 4 grade III). Complete response was obtained in 7 cases; 2 patients had a partial response and finally 1 was a nonresponder and showed local progression, which required surgical amputation. Tumor necrosis (observed in 5 cases) was maximal in 4 patients (80-100%) and absent in the patient who had local progression. The results we obtained with the treatment of soft tissue sarcoma confirm the efficacy of ILP as a limb-sparing methodology for unresectable recurrences. Furthermore, rHu TNF-alpha and melphalan confirmed their antitumor activity when associated with hyperthermia. Amputation or disarticulation may be reliable as a second-choice treatment for these patients.

Effects of tumour necrosis factor ? on bone marrow aspirates of patients with acute myelogenous leukemia determined by flow-cytometric cell-cycle analysis

Tumour necrosis factor alpha (TNF alpha) exerts cytotoxic and antiproliferative effects on neoplastic cells. It has been used as a therapeutic agent for solid tumours and haematological malignancies. We report on the ex vivo determination of the effect of recombinant human rhuTNF alpha on bone marrow aspirates by a bromodeoxyuridine/propidium iodide method. Cell samples were drawn after 0.5, 2, 4, 6, 8, 10, 22, and 25 h from short-term suspension bone marrow cultures from patients with acute myelogenous leukemia (AML). Flow-cytometric cell-cycle analysis was performed after double DNA staining with propidium iodide and anti-BrdU antibodies. By this method the effect of rhuTNF alpha on cell proliferation can be evaluated after only 35 h. In about two-thirds of the bone marrow aspirates of AML an inhibiting effect on rhuTNF alpha can be demonstrated, developing to its full extent after 10 h.

Effects of recombinant tumor necrosis factor (rHuTNFα)on human neutrophils and monocytes: In vitro, ex vivo and in vivo

In in vitro experiments, tumor necrosis factor (rHuTNF alpha) was found to inhibit spontaneous and directed migration of normal human neutrophils and monocytes. RHuTNF alpha showed no chemoattractant activity. In conjunction with a Phase-2 clinical trial, we also studied in vivo rHuTNF alpha effects on neutrophil and monocyte number and function. At the time of maximal plasma TNF levels (30 min), marked neutropenia and monocytopenia were observed. Isolated neutrophils were activated for superoxide production but were unable to migrate. Monocyte migration was inhibited at later times. Neutrophil migratory function recovered between 4 and 8 h but was again depressed at 24 h. The data demonstrate the complexity of the response to TNF, comprising direct and indirect effects which are concentration-, time- and place-dependent. They further suggest that the only neutrophils and monocytes available for participation in an anti-tumor activity of TNF in vivo are those present in the tumor at the outset.

Some recombinant human cytokines stimulate glycosaminoglycan synthesis in human synovial fibroblast cultures and inhibit it in human articular cartilage cultures

Recombinant human cytokines were compared for their effects on glycosaminoglycan (GAG) synthesis in human synovial fibroblast cultures and human articular cartilage explant cultures. In fibroblast cultures, recombinant human interleukin-1 alpha (rHuIL-1 alpha), rHuIL-1 beta, and recombinant human tumor necrosis factor alpha (rHuTNF alpha) stimulated hyaluronic acid (HA) production and, to a lesser extent, sulfated GAG production, while recombinant human gamma-interferon did not have a significant effect. Half-maximal stimulation of HA by rHuIL-1 beta was 0.14 pM, while stimulation for rHuIL-1 alpha and rHuTNF alpha was 1.6 pM and 32 pM, respectively. Indomethacin (10 micrograms/ml) had no influence on HA stimulation by cytokines, while hydrocortisone (2-10 micrograms/ml) caused a significant reduction. In articular cartilage cultures, the cytokines inhibited production of sulfated GAGs. The activity of rHuIL-1 beta was greater than that of rHuIL-1 alpha (half-maximal inhibition at 0.71 pM and 4.7 pM, respectively) and both were considerably more active than rHuTNF alpha; gamma-interferon again had no significant effect. Neither indomethacin nor hydrocortisone influenced cytokine-induced inhibition by either rHuIL-1 preparation. These studies indicate that cytokines released during an inflammatory process may affect GAG synthesis in human joint tissues and may have opposite effects on GAG synthesis in different types of connective tissues.

Induction of Dermal Subcutaneous Inflammation by Recombinant Cachectin/Tumor Necrosis Factor (TNFα) in the Mouse

The ability of cachectin/tumor necrosis factor (TNF alpha) to induce acute dermal and subcutaneous inflammation was examined in a murine model. A number of other proteins, and diluent alone were examined as controls. After subcutaneous injection into the mouse footpad, recombinant human TNF alpha (rHuTNF alpha) induced acute inflammation with an initial marked dermal and subcutaneous neutrophil infiltrate by approximately 3 h, with a peak between 4 and 24 h and resolution by 79 h. Recombinant interleukin-2, cytochrome c, and heat-inactivated rHuTNF alpha induced negligible inflammation. Recombinant human lymphotoxin (TNF beta), another control protein, also induced acute inflammation in our system. Because TNF alpha and TFN beta are partially homologous, they may be acting through a similar mechanism. This pro-inflammatory effect of TNF alpha may result from chemotactic activity as well as by induction of secondary mediators. Inflammation induced by TNF alpha was partially suppressed by indomethacin treatment, suggesting that products of the cyclo-oxyganase pathway may mediate a portion of the inflammation involved. Five daily injections of rHuTNF alpha into the mouse footpad resulted in a predominantly mononuclear infiltrate and focal fibrosis. These results suggest that TNF alpha may be an important mediator of acute inflammation in vivo and might provide a signal for the production of collagen.

Recombinant human tumor necrosis factor α lacks chemotactic activity for human peripheral blood neutrophils and monocytes

Preparations of recombinant human tumor necrosis factor alpha (rhuTNF alpha) free of aminoterminal methionine were tested for human neutrophil granulocyte (PMN) and monocyte (MO) chemotactic activity using the Boyden chamber system. Over a wide range of concentrations (10(-7)-10(-15) M) rhuTNF alpha of two different sources failed to elicit chemotactic responses in PMN or MO, whereas strong PMN and MO chemotactic activity could be detected using the tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). In addition, rhuTNF alpha containing 62% aminoterminal methionine failed to induce PMN and MO chemotaxis. It is concluded that rhuTNF alpha may not be a chemotaxin for human PMN and MO in vitro.

Tumor Necrosis Factor Causes Increased Pulmonary Permeability and Edema: Comparison to Septic Acute Lung Injury

Tumor necrosis factor alpha (TNF), a monokine produced by mononuclear cells in response to bacterial endotoxin (LPS), creates a syndrome similar to septic shock in animal models. To study whether TNF could induce acute lung injury similar to that seen in gram-negative sepsis, we injected recombinant human TNF (rHuTNF alpha) into guinea pigs and monitored arterial blood gases, leukocyte counts, and left atrial (Pla), pulmonary artery (Ppa), and mean arterial pressures (MAP) serially for 8 h. Pulmonary histopathology was assessed microscopically, and cell counts and 125I-labeled albumin (125I-albumin) in bronchoalveolar lavage (BAL) fluid and lung wet/dry weight ratios were determined. Five groups of animals were studied; the 2 TNF groups received high (1.4 X 10(6) U/kg) or low (1.0 X 10(6) U/kg) doses of rHuTNF alpha, the sepsis group received 2 X 10(9) Escherichia coli/kg intravenously, and the control group received saline. An LPS control group receiving 40 ng/kg E. coli LPS was also included because the rHuTNF alpha contained a small amount of LPS as a contaminant. Pulmonary permeability was assessed by studying the Pla and the BAL fluid/plasma 125I-albumin ratio (permeability index). The permeability index was significantly increased in the high-dose TNF (0.0408 +/- 0.0041, p less than 0.05) and sepsis groups (0.0466 +/- 0.0068, p less than 0.01) relative to controls (0.0215 +/- 0.0028). The wet/dry lung weight ratios were also significantly increased in the high-dose TNF (6.07 +/- 0.29, p less than 0.05) and sepsis groups (6.22 +/- 0.30, p less than 0.05) relative to the control group (5.18 +/- 0.20).(ABSTRACT TRUNCATED AT 250 WORDS)