Eva M. Horak

The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck

The CD4 and CD8 T cell antigens are thought to transduce an independent signal during the process of T cell activation. We report our evaluation of the possible involvement of the lymphocyte-specific tyrosine kinase p56lck in these transduction pathways. Our data demonstrate that p56lck is specifically modulated with either CD4 or CD8 following antibody-mediated cross-linking of these molecules and that a large fraction of the total cellular lck protein can be coimmunoprecipitated with these surface glycoproteins. These results suggest that p56lck is functionally and physically associated with CD4/CD8 in normal murine T lymphocytes and support the concept that an independent signal is transduced by the interaction of these surface molecules with major histocompatibility complex determinants.

Increased concentrations of cholestanol and apolipoprotein B in the cerebrospinal fluid of patients with cerebrotendinous xanthomatosis. Effect of chenodeoxycholic acid.

We investigated the effect of chenodeoxycholic acid on cerebrospinal fluid sterol and protein composition in six patients with cerebrotendinous xanthomatosis, a progressive neurologic disease, and in 11 control subjects. In the cerebrospinal fluid from the controls, the mean (+/- SD) levels of cholesterol and cholestanol were 400 +/- 300 and 4 +/- 7 micrograms per deciliter, respectively. The levels were almost 1.5 and 20 times higher in cerebrospinal fluid from untreated patients with cerebrotendinous xanthomatosis. Cholestanol levels were also markedly elevated in the plasma of untreated patients, but their plasma cholesterol levels (215 +/- 61 mg per deciliter) were not different from control values. Treatment with chenodeoxycholic acid reduced cerebrospinal fluid cholesterol by 34 percent and cholestanol threefold. Plasma cholestanol levels also decreased sharply. Normal cerebrospinal fluid contained small quantities of albumin, apolipoproteins, and lecithin:cholesterol acyltransferase. In cerebrospinal fluid from untreated patients with cerebrotendinous xanthomatosis, immunoreactive apolipoprotein B or apolipoprotein B fragment was increased about 100-fold and albumin about 3.5-fold; apolipoprotein AI, apolipoprotein D, and lecithin:cholesterol acyltransferase were 1.5 to 3 times more concentrated. Apolipoprotein AIV and apolipoprotein E concentrations were comparable to those in controls, and apolipoprotein AII was considerably decreased. During treatment, the concentrations of albumin and apolipoproteins AI and B declined. These results suggest that increased cerebrospinal fluid sterols are derived from plasma lipoproteins by means of a defective blood-brain barrier in patients with cerebrotendinous xanthomatosis. Therapy with chenodeoxycholic acid reestablished selective permeability of the blood-brain barrier and normalized the concentrations of sterol and apolipoprotein in the cerebrospinal fluid.

Effects of the tyrosine‐kinase inhibitor geldanamycin on ligand‐induced HER‐2/NEU activation, receptor expression and proliferation of HER‐2‐positive malignant cell lines

Geldanamycin belongs to the family of benzoquinoid ansamycin tyrosine‐kinase inhibitors. We have examined its effects on Her‐2/neu kinase activity, protein expression level, and proliferation of Her‐2+ malignant cells. In SK‐BR‐3 breast‐cancer cells, short‐time treatment with geldanamycin completely abrogated gp30‐ligand‐induced activation of Her‐2 without a change of receptor‐expression level. Longer treatment of intact cells with geldanamycin induced decreased steady‐state Her‐2 autophosphorylation activity, which correlated with reduction of Her‐2 protein expression and phosphotyrosine content of several proteins. The decrease was time‐ and dose‐dependent, starting after 1 hr at 100 nM concentration and reaching completion by 24 hr. The reduction of the Her‐2 protein level probably resulted from increased degradation, since the Her‐2 mRNA level remained constant. Geldanamycin effects were not specific for Her‐2, since the non‐receptor tyrosine‐kinase fyn was inhibited equally. In contrast to these results, protein‐kinase‐C activity was not affected. In 3 other malignant cell lines expressing different amounts of Her‐2 (SK‐BR‐3 > SK‐OV‐3 > OVCAR3 > MCF7), geldanamycin also effectively reduced Her‐2‐kinase activity proportionally to the decrease of protein expression. In contrast, in a [3H]‐thymidine‐uptake assay, cell growth was meaningfully inhibited by geldanamycin at nanomolar concentrations only in SK‐BR‐3 (IC50 2nM) and MCF7 (IC50 20nM), while OVCAR3 was only moderately sensitive (IC50 2μM) and SK‐OV‐3 was clearly resistant to geldanamycin. In direct comparison with herbimycin A, another benzoquinoid ansamycin that has been more thoroughly characterized, the biologic effects of geldanamycin were more pronounced. Int. J. Cancer, 70:221–229, 1997.

Preparation and in vivo evaluation of linkers for 211At labeling of humanized anti-Tac

The syntheses, radiolabeling, antibody conjugation, and in vivo evaluation of new linkers for 211At labeling of humanized anti-Tac (Hu-anti-Tac), an antibody to the alpha-chain of the IL-2 receptor (IL-2Ralpha) shown to be a useful target for radioimmunotherapy are described. Synthesis of the organometallic linker precursors is accomplished by reaction of the corresponding bromo- or iodoaryl esters with bis(tributyltin) in the presence of a palladium catalyst. Subsequent conversion to the corresponding N-succinimidyl ester and labeling with 211At of two new linkers, N-succinimidyl 4-[211At]astato-3-methylbenzoate and N-succinimidyl N-(4-[211At]astatophenethyl)succinamate (SAPS), together with the previously reported N-succinimidyl 4-[211At]astatobenzoate and N-succinimidyl 3-[211At]astato-4-methylbenzoate, are each conjugated to Hu-anti-Tac. The plasma survival times of these conjugates are compared to those of directly iodinated (125I) Hu-anti-Tac. The N-succinimidyl N-(4-[211At]astatophenethyl)succinamate compound (SAPS) emerged from this assay as the most viable candidate for 211At-labeling of Hu-anti-Tac. SAPS, along with the directly analogous radio-iodinated reagent, N-succinimidyl N-(4-[125I]astatophenethyl)succinamate (SIPS), are evaluated in a biodistribution study along with directly iodinated (125I) Hu-anti-Tac. Blood clearance and biological accretion results indicate that SAPS is a viable candidate for further evaluation for radioimmunotherapy of cancer.