Dendritic cells from patients with myeloma are numerically normal but functionally defective as they fail to up-regulate CD80 (B7-1) expression after huCD40LT stimulation because of inhibition by transforming growth factor-beta1 and interleukin-10.

Limited response to idiotype vaccination in patients with myeloma suggests that there is a need to develop better immunotherapy strategies. It has been determined that the number of high-potency CMRF44+CD14-CD19- dendritic cells (DCs) in the blood of patients with myeloma (range, 0.03%-0.8% of mononuclear cells [MNCs]; n = 26) was not significantly different from that in controls (range, 0.05%-0.8% of MNCs; n = 13). Expression of the costimulatory molecules CD80 and CD86 on DCs from these patients (mean, 29%+/-17% of MNCs and 85%+/-10% of MNCs, respectively) was also normal (mean, 29%+/-17% and 86%+/-16% of MNCs, respectively). Up-regulation of CD80 expression in response to stimulation by human huCD40LT + interleukin (IL)-2 was significantly reduced on the DCs of patients with myeloma during stable disease (n = 9) and was absent during progressive stages (n = 7) of disease. Similar effects were seen on B cells but not on monocytes of the same group of patients. CD86 expression on DCs was high before (86%) and after (89%) stimulation. Inhibition of CD80 up-regulation was neutralized by either anti-transforming growth factor (TGF)-beta1 or anti-IL-10. Up-regulation of CD80 on DCs of controls was inhibited by rTGF-beta1 in a dose-dependent manner. Serum TGF-beta1 and IL-10 levels were normal in most patients studied. Cytoplasmic TGF-beta1 was increased in plasma cells during progressive disease. Thus patients with myeloma have normal numbers of DCs, but CD80 expression may fail to be up-regulated in the presence of huCD40LT because of tumor-derived TGF-beta1 or IL-10. Autologous high-potency DCs may have to be tested for CD80 up-regulation and biologically modified ex vivo before idiotype priming for immunotherapy.

T-cell expansions in patients with multiple myeloma have a phenotype of cytotoxic T cells.

The presence of T-cell clones in peripheral blood has been previously shown to be associated with a survival advantage in patients with multiple myeloma and suggests that the expanded T-cell populations may be involved in an anti-tumour response. We studied the T-cell receptor (TCR) repertoire of 38 patients with myeloma to identify and characterize the expanded T-cell populations by flow cytometry. T-cell expansions were found in 79% of the patients. The expansions occurred randomly among the 21 variable regions of the TCR beta chain (Vbeta) studied, representing 62% of the V-beta repertoire, and were stable during an 18-month follow-up. The phenotype of the expanded V-beta populations was predominantly CD8+, CD57+, CD28- and perforin+, which differed significantly from the other non-expanded Vbeta populations. The expression of the apoptosis markers Fas (CD95) and bcl-2 were similar between the expanded and non-expanded Vbeta populations. In conclusion, expanded T-cell populations were frequent in patients with myeloma, they remained unchanged during follow-up and had phenotypic characteristics of cytotoxic T cells. These data add further support to the concept that the T-cell expansions may have an immunoregulatory role in myeloma.

Preparation, characterization, DNA binding, and in vitro cytotoxicity of the enantiomers of the platinum(II) complexes N-methyl-, N-ethyl- and N,N-dimethyl-(R)- and -(S)-3-aminohexahydroazepinedichloroplatinum(II).

A series of chiral diaminedichloroplatinum(II) complexes derived from [Pt(ahaz)Cl2] (ahaz = 3-aminohexahydroazepine) have been synthesized and tested for cytotoxic activity. Novel synthetic pathways were developed to produce the structural derivatives of the ahaz ligand, with alkyl substituents on the exocyclic nitrogen atom. The platinum(II) complexes of these ligands were synthesized and characterized by NMR and CD spectroscopy, confirming isomeric and enantiomeric purity. The crystal structures of three of these complexes, [Pt(S-meahaz)-Cl2], [Pt(R-etahaz)Cl2], and [Pt(S-dimeahaz)Cl2] (meahaz = N-methylahaz, etahaz = N-ethylahaz, dimeahaz = N,N-dimethylahaz), have been determined to establish the orientation of the protons and alkyl substituents on the nitrogen donor atoms. In vitro assays of the cytotoxic activity of the complexes have revealed a significant and reproducible enantioselective trend with the R-enantiomers more active than the S-enantiomers in all cell lines. Increasing the steric bulk on the amine groups was found to have only a modest effect on activity. No enantioselectivity was observed in the binding of R- and S-[Pt(etahaz)Cl2] to calf-thymus DNA.

The efficacy of doxorubicin microspheres for hepatic micrometastases in a rat tumour model.

The use of sustained-release microspheres is of potential benefit as an adjuvant treatment for patients with occult hepatic micrometastases. This study investigates the response of a model of implantable adenocarcinoma micrometastases in the livers of DA rats following the intraportal injection of doxorubicin-incorporated ion-exchange microspheres compared to free drug bolus administration. A point-counting technique was used to determine the percentage of liver consisting of tumour 13 days after treatment. This was used as an indicator of tumour response, as was the derived tumour mass. There was a significantly higher tumour response in animals treated with the microspheres compared to animals treated with free drug delivered at the same concentration. This effect, however, was shown to decrease with a delay in the time of treatment. The tumour response of the sustained-release microspheres was achieved in the absence of any detectable local or systemic toxicity. This study demonstrates the potential of sustained-release microspheres in the treatment of patients with hepatic micrometastases.

Tumour cells surviving in vivo cisplatin chemotherapy display elevated c-myc expression.

The c-myc oncogene has been extensively implicated in cell proliferation, cell differentiation and programmed cell death. Aberrant expression of the c-myc gene product has been observed in a range of tumours and has also been implicated in cisplatin (cis-dichlorodiammineplatinum)-mediated chemoresistance. A solid transplantable tumour model in syngeneic DA rats was subjected to treatment with cisplatin to determine the impact of such therapy on endogenous c-myc gene expression. Serially transplanted tumours were intravenously treated with a single cisplatin dose (1 mg/kg) and c-myc expression analysed 2 and 7 days after treatment. The surviving tumour cells display a significant 2-fold elevation in c-myc expression at 48 h and 7 days after treatment. Primary cell cultures have been derived from untreated in vivo tumours of the same model and subjected to treatment with a c-myc phosphorothioate antisense oligomer. Administration of 5 microM c-myc antisense oligomer directed at the initiation codon and first four codons of c-myc mRNA results in total inhibition of c-myc expression and coincident suspension of cell growth for a period of 4 days in culture. Antisense therapies directed at the c-myc gene may well prove an effective tool for treating tumours in conjunction with cisplatin as these findings show that tumour cells surviving cisplatin chemotherapy display elevated c-myc expression.

In vitro lactate metabolism by ruminal ingesta.

Ruminal ingesta (300 ml) obtained from a fistulated cow fed alfalfa hay (H), 3.6 kg of grain mixture with corn silage fed ad libitum (S), 2.5:1 grain-alfalfa hay mixture (G), or a 2.5:1 grain-alfalfa hay mixture providing 545 g of sodium and calcium lactate daily (L) were incubated for 8 hr with nonpolymerized sodium lactate or 17% polymerized lactic acid neutralized to pH 6.7. Polymerization had no effect on the rate of lactate utilization. The initial rates of lactate metabolism for the H, G, S, and L ingesta were 0.72, 0.95, 1.8, and 3.4 meq per 100 ml of rumen fluid per hr, respectively. Lactate-2-(14)C was incubated for 4 hr with each type of ruminal ingesta. Of the label recovered in the volatile fatty acids (VFA), 74.1, 61.2, 49.3, and 38.9% was recovered in acetate, and 9.4, 19.8, 23.3, and 51.9% was recovered in propionate with H, G, S, and L ingesta, respectively. The balance of label was distributed between butyrate and valerate. The titratable VFA did not follow this pattern of production. With the hay ingesta, lactate metabolism resulted in a net loss of acetate and a large increase in butyrate. Little propionate was produced. The G, S, and L ingesta metabolized lactate to yield progressively more propionate and less butyrate. Evidence was gathered to suggest that acetate was the primary end product of lactate metabolism but that oxidation of lactate to pyruvate dictated the synthesis of butyrate from acetate to maintain an oxidation-reduction balance. It was noted that acetate and butyrate production from lactate was pH-dependent, with acetate production maximal at pH 7.4 and butyrate at 6.2. Propionate production was largely unaffected within this pH range.