Ring and nodular multiple sclerosis lesions: a retrospective natural history study.

BACKGROUND: In patients with multiple sclerosis (MS), contrast-enhancing lesions (CELs) on postcontrast MRI are considered markers of the inflammatory responses associated with blood-brain barrier breakdown. Based upon shape, CELs may be defined as nodular (nCEL) or ring (rCEL) lesions. Several short-term studies pointed towards the assumption that rCELs represent areas of a more aggressive inflammatory process. METHODS: In the present long-term (i.e., 2 years) retrospective natural history study, we used monthly imaging to follow rCEL and nCELs evolution in 16 patients with MS during the natural history. New CELs were identified monthly on month 4-9 MRIs, using month 1-3 MRIs to ensure that all CELs were not previously enhancing. Chronic black holes (cBHs) were counted monthly upon CEL disappearance up to the 24th MRI. Generalized estimating equation methods investigated within-patient differences between rCELs and nCELs in volume and likelihood to convert into cBHs. Kaplan-Meier survival curves estimated differences in the length of persistence between cBHs originating from nCELs and cBHs deriving from rCELs. RESULTS: Fifty-two new rCELs and 281 nCELs were identified. rCELs had larger mean (z = 5.06, p < or = 0.0001) volumes than nCELs. The proportion of cBHs from rCELs was similar (z = 1.81, p = 0.0710) to the proportion of cBHs from nCELs. Likewise, the length of persistence of cBHs deriving from rCELs was similar (chi(1)(2) = 2.339, p = 0.1262) to the duration of cBHs from nCELs. CONCLUSIONS: Our data suggest that worse radiologic characteristics associated with the acute phase of ring contrast-enhancing lesions and nodular contrast-enhancing lesions do not necessarily reflect a poorer lesion outcome over time.

Tissue-specific imaging is a robust methodology to differentiate in vivo T1 black holes with advanced multiple sclerosis-induced damage.

BACKGROUND AND PURPOSE: Brains of patients with multiple sclerosis (MS) characteristically have "black holes" (BHs), hypointense lesions on T1-weighted (T1W) spin-echo (SE) images. Although conventional MR imaging can disclose chronic BHs (CBHs), it cannot stage the degree of their pathologic condition. Tissue-specific imaging (TSI), a recently introduced MR imaging technique, allows selective visualization of white matter (WM), gray matter (GM), and CSF on the basis of T1 values of classes of tissue. We investigated the ability of TSI-CSF to separate CBHs with longer T1 values, which likely represent lesions containing higher levels of destruction and unbound water. MATERIALS AND METHODS: Eighteen patients with MS, who had already undergone MR imaging twice (24 months apart) on a 1.5T scanner, underwent a 3T MR imaging examination. Images acquired at 1.5T included sequences of precontrast and postcontrast T1W SE, T2-weighted (T2W) SE, and magnetization transfer (MT). Sequences obtained at 3T included precontrast and postcontrast T1W SE, T2W SE, T1 inversion recovery prepared fast spoiled gradient recalled-echo (IR-FSPGR) and TSI. A BH on the 3T-IR-FSPGR was defined as a CBH if seen as a hypointense, nonenhancing lesion with a corresponding T2 abnormality for at least 24 months. CBHs were separated into 2 groups: those visible as hyperintensities on TSI-CSF (group A), and those not appearing on the TSI-CSF (group B). RESULTS: Mean MT ratios of group-A lesions (0.22 +/- 0.06, 0.13-0.35) were lower (F(1,13) = 60.39; P < .0001) than those of group-B lesions (0.32 +/- 0.03, 0.27-0.36). CONCLUSIONS: Group-A lesions had more advanced tissue damage; thus, TSI is a potentially valuable method for qualitative and objective identification.

Thalamic involvement and its impact on clinical disability in patients with multiple sclerosis: a diffusion tensor imaging study at 3T.

BACKGROUND AND PURPOSE: Several studies suggest that grey matter involvement may play a role in multiple sclerosis (MS) pathology. Diffusion tensor imaging (DTI) at 3T was used to investigate the presence of damage to the normal-appearing thalamus in MS and its relationship with disability. MATERIALS AND METHODS: Twenty-four patients with relapsing-remitting (RR, n = 13, age = 41.7 +/- 6.1, Expanded Disability Status Scale [EDSS] score = 2.2 +/- 1.2) and secondary-progressive (n = 11, age = 46.9 +/- 9.6, EDSS = 5.9 +/- 1.0) MS and 24 age- and sex-matched healthy volunteers were studied. Fractional anisotropy (FA) and mean diffusivity (MD) were measured in regions of interest of normal-appearing thalamus. We examined group differences in MD and FA and correlations between DTI-derived metrics and clinical or imaging measures of disease. RESULTS: Patients with MS had higher thalamic FA (P < .0001) and MD (P = .035) than volunteers. MD values correlated with the Paced Auditory Serial Addition Task (r = -0.43, P = .034) and motor EDSS (r = 0.47, P = .021) scores. In patients with RRMS, MD values correlated with global EDSS (r = 0.75, P = .003) and motor EDSS (r = 0.68, P = .010). Correlations were found between MD values and T1 and T2 lesion load (r = 0.58, P < .05) and brain parenchymal fraction (r = -0.46, P < .05). CONCLUSIONS: DTI was able to detect abnormalities in normal-appearing thalamus of patients with MS. The strength of association between thalamic DTI measures and functional impairment was in the same range as those seen with standard MR imaging disease measures. The assessment of the integrity of the thalamus with DTI is a promising metric as a marker of disease for future studies.

A case study on the effect of neutralizing antibodies to interferon beta 1b in multiple sclerosis patients followed for 3 years with monthly imaging.

Interferon beta (IFN-beta) is among the first-line treatment options for patients with multiple sclerosis (MS). A potential caveat of therapy, however, is the development of neutralizing antibodies (NAb) and/or neutralizing activity (NA) non-antibody mediated, although debate is still ongoing as to whether NAb significantly hampers the efficacy of the drug or rather represents an immunologically irrelevant epiphenomenon. In the present study, we describe the effect of NAb on IFN-beta-1b through clinical and magnetic resonance imaging (MRI) outcome measures of five relapsing-remitting multiple sclerosis (RRMS) patients who were treated with 250 mug of subcutaneously administered IFN-beta-1b every other day and developed NAb at varying titres and times during the course of therapy. Despite the small number of NAb(+) patients, heterogeneity in MRI/clinical response to IFN-beta-1b was identified. Response to IFN-beta-1b therapy was observed in the absence or presence of NAb. Also observed was failure to IFN-beta-1b coincident with high and sustained NAb titres, but also before NAb development or in the presence of low NAb titres. Multiple MRI and NAb measurements performed within the same individual allow for a better description of the complex heterogeneous response to IFN-beta-1b with respect to NAb occurrence.

Relationship between inflammatory lesions and cerebral atrophy in multiple sclerosis.

OBJECTIVE: To investigate the temporal relationship between inflammation and cerebral atrophy in a longitudinal study of 19 patients with relapsing-remitting multiple sclerosis (RRMS) using serial monthly contrast enhanced MRI examinations and monthly measurements of brain fractional volume (BFV) for an average of 4 (range 2.4 to 10) years. METHODS: In this retrospective study, all patients had an active MRI scan at entry with a minimum of two new contrast enhancing lesions (CEL) on baseline MRI examinations. Patients were followed for a minimum of 3 months during a baseline (pretreatment) phase and subsequently followed during treatment with recombinant interferon beta (IFN) and various other immunomodulatory agents. Pre- and post contrast axial images were obtained using 3-mm slice thickness and a gadolinium contrast dose of 0.1 mmol/kg. Monthly CEL were sequentially numbered on hardcopy films and monthly BFV was determined on precontrast T1W images using a semiautomated program. For BFV measurements, all T1W scans were registered to the entry examination, which served as a mask image. Cerebral atrophy was measured as percent brain fractional volume change (PBVC) compared to the entry baseline scan. RESULTS: The results demonstrate that cerebral atrophy paralleled that of contrast enhancing lesion accumulation. The correlation between cumulative CEL and PBVC ranged from R2 = 0.47 to 0.81. Immunomodulatory agents that effectively reduced CEL accumulation also slowed the rate of atrophy. CONCLUSIONS: The correlation between contrast enhancing lesions (CEL) and atrophy suggests that patients who are not responding to therapy with a decrease in CEL may also be at risk for developing increased atrophy.

Interferon-beta-1b effects on re-enhancing lesions in patients with multiple sclerosis.

Interferon-beta (IFNbeta) reduces the number and load of new contrast-enhancing lesions (CELs) in patients with multiple sclerosis (MS). However, the ability of IFNbeta to reduce lesion sizes and re-enhancements of pre-existing CELs has not been examined extensively. Activity of contrast re-enhancing lesions (Re-CELs) and contrast single-enhancing lesions (S-CELs) were monitored in ten patients with relapsing-remitting (RR) MS. These patients underwent monthly post-contrast magnetic resonance imaging (MRIs) for an 18-month natural history phase and an 18-month therapy phase with subcutaneous IFNbeta-1b, totaling 37 images per patient. The activity was analysed using the first image as a baseline and registering subsequent active monthly images to the baseline. There was a 76.4% reduction in the number of CELs with IFNbeta therapy. The decrease was greater (P = 0.003) for S-CELs (82.3%) than for Re-CELs (57.4%). S-CELs showed no changes in durations of enhancement and maximal lesion sizes with treatment. Exclusively for Re-CELs, IFNbeta-1b significantly decreased maximal lesion sizes, total number of enhancement periods and total months of enhancement. Thus, IFNbeta appears to be effective in reducing the degree of severity of inflammation among Re-CELs, as reflected by their reduced maximal lesion sizes and durations of enhancement.

Contrast-enhanced MRI lesions during treatment with interferonbeta-1b predict increase in T1 black hole volume in patients with relapsing-remitting multiple sclerosis.

T1 black holes (BH) have been found to represent focal areas of substantial central nervous system tissue damage in multiple sclerosis (MS) patients. We examined the development of T1 BH over a three-year period of treatment with interferon (IFN)beta-1b in a group of 20 patients with relapsing-remitting MS. The number of contrast-enhancing lesions (CEL) after one year of treatment predicted a change in the T1 BH volume in the following two years. In patients without CEL, the T1 BH volume remained stable, whereas it increased in patients with CEL. The occurrence of CEL in patients treated with IFNbeta may indicate a heightened risk of accumulating T1 BH.

Ring-enchancement in multiple sclerosis: marker of disease severity.

Correlations between conventional MRI measures of disease activity and clinical disability in multiple sclerosis (MS) have been disappointing. Because ring-enhancing lesions may reflect a more destructive pathology, we tested their potential association with disease severity. We evaluated active lesions with regard to their enhancement pattern on serial magnetic resonance images in a cohort of 28 patients with relapsing-remitting MS. The percentage of ring-enhancing lesions correlated with EDSS, T2 lesion load and duration of disease and predicted the occurrence of relapses during the baseline period of observation as well as after 3 years of follow-up in multiple logistic regression analysis. The findings suggest that the pathological process reflected by ring-enhancing lesions may contribute to more severe clinical disease.

Interferon beta-1b and intravenous methylprednisolone promote lesion recovery in multiple sclerosis.

OBJECTIVE: To determine whether lesion evolution in relapsing-remitting multiple sclerosis (RRMS) patients is altered by treatment with interferonbeta1b (IFNbeta-1b) or by intravenous methylprednisolone (IVMP) as measured by magnetization transfer imaging. METHODS: Magnetization transfer ratios (MTR) of 225 contrast enhancing lesions (CEL), in four RRMS patients were serially determined for 12 months before and 12-18 months after contrast enhancement in a baseline vs treatment trial with IFNbeta-1b. During the baseline period, 185 new CEL were identified: 76 were treated with IVMP (1 g/day x 5 days) and designated steroid CEL (S-CEL); the remaining 109 were considered baseline lesions (BCEL). During IFNbeta-1b treatment, 40 CEL (IFN-CEL) were identified. After image co-registration, regions of interest (ROIs) defining new CEL were transferred to the MTR image set to determine the mean lesion MTR on each monthly exam. The lesion MTR was compared to MTR of normal appearing white matter (NAWM) on the same exam. RESULTS: As early as 12 months prior to enhancement, the MTR of CEL was reduced compared to NAWM (mean 9.43 +/- 3.2%; P<0.001). The further reduction in MTR (28% +/- 4.0) at the time of contrast enhancement was not significantly different for BCEL, S-CEL or IFN-CEL Following enhancement, lesion recovery for IFN-CEL (P=0.02) and S-CEL (P=0.002) was significantly higher than BCEL CONCLUSION: IFNbeta-1b and IVMP reduce tissue damage and promote lesion recovery in RRMS patients. The additional benefit of IVMP compared to IFNbeta-1b may be related to its inhibitory effect on demyelination.

MRI and clinical activity in MS patients after terminating treatment with interferon beta-1b.

Monthly MRI activity and clinical disability were evaluated in two relapsing-remitting multiple sclerosis (RRMS) patients for 4 years during a cross-over treatment trial with IFNbeta-1b, and for a mean of 21 months after terminating treatment with IFNbeta-1b. Post-treatment MRI activity was compared to baseline activity in these patients. Although contrast enhancing lesions (CEL) and the bulk white matter lesion load (BWMLL) on T2-weighted images eventually returned to baseline values, there was a refractory period of 6 - 10 months after terminating treatment, before baseline MRI activity was restored. Although the mechanism for a sustained effect of IFNbeta-1b is unclear at this time, these results have important implications for enrollment of such patients into new treatment protocols that rely on contrast enhancing lesion frequency as an outcome measure.

Effect of open label pulse cyclophosphamide therapy on MRI measures of disease activity in five patients with refractory relapsing-remitting multiple sclerosis.

OBJECTIVE: To evaluate the response to cyclophosphamide (CTX) of five patients who failed an average three treatments with multiple other therapeutic agents, using serial monthly MRI measures. METHODS: Five patients with relapsing-remitting multiple sclerosis (MS) and documented MRI disease activity were started on monthly pulse intravenous CTX at a dose of 1 g/m2. CTX was administered without an induction phase according to the protocol similar to the treatment of lupus nephritis. The five patients were followed with monthly MRI and clinical evaluation for a mean of 28 months. RESULTS: All the patients showed a rapid reduction in the contrast-enhancing lesion frequency and in three patients there was a decrease in the T2 lesion load within the first 5 months after starting CTX treatment. The administration of CTX during overnight hospitalization was safe and well tolerated. CONCLUSIONS: These findings suggest that aggressive immunosuppressive therapy may be useful in some rapidly deteriorating refractory patients and further controlled study should be considered in order to full evaluate this type of treatment as a potential therapy in MS.

Magnetization transfer imaging to monitor clinical trials in multiple sclerosis.

Magnetization transfer imaging (MTI) is a sensitive magnetic resonance scanning technique for detecting disease activity and monitoring disease progression in multiple sclerosis (MS) patients. To date, MTI has not been used as an outcome measure in early phase and pivotal clinical trials. Magnetization transfer ratio, a quantitative measure of MT, can be used to follow the evolution of individual MS plaques as well as microscopic disease in normal-appearing white matter (NAWM). Volumetric whole brain MT ratios of every voxel in the brain can be expressed as a histogram, providing an estimate of cerebral atrophy and a global disease activity including "occult" disease in NAWM, thus providing a quantitative measure of a therapeutic response. This report will briefly focus on how MTI could be used to monitor a therapeutic response in a treatment trial and address some of the pitfalls/limitations of MTI that can be encountered even in a well-designed longitudinal study.

Characterization of differences between multiple sclerosis and normal brain: a global magnetization transfer application.

BACKGROUND AND PURPOSE: Although the exact nature of the physiological differences between normal and multiple sclerosis (MS) brains are unknown, it has been shown that their global magnetization transfer ratio (MTR) values are significantly different. To more fully understand these differences, we examined MTR values by using 30 distinct measures. We provide a unique illustration of these differences through a derived normal-to-MS transform. METHODS: Global MTR values for the group of normal subjects and for the group of MS subjects were characterized by 30 different measures involving simple statistics, histographic characteristics, MTR order information, and MTR range information. The measures that were significantly different with respect to these two groups were discovered. From the mean MTR histogram of the two groups, a transform was created to describe a conversion between the two brain states. Normal data were passed through this transform, creating a set of pseudo-MS data. The measures that were significantly different from the normal and pseudo-MS data were also obtained in order to verify the accuracy of the transform. RESULTS: Seventeen of the 30 measures were determined to be significantly different when comparing the sets of normal and MS data. The same set of 17 measures were found to be significantly different when comparing the normal and pseudo-MS data. CONCLUSION: The differences in the global MTR values of normal and MS subjects are statistically significant compared with a large number of measures (alpha = 0.05). A normal-to-MS transform is a novel method for illustrating these differences.

Serial whole-brain magnetization transfer imaging in patients with relapsing-remitting multiple sclerosis at baseline and during treatment with interferon beta-1b.

BACKGROUND AND PURPOSE: To determine whether occult disease fluctuates with macroscopic lesions during the natural history of multiple sclerosis (MS) and whether therapeutic interventions affect occult disease, we performed serial monthly magnetization transfer (MT) imaging in patients with relapsing-remitting MS in a crossover trial with interferon beta-lb. METHODS: Serial whole-brain magnetization transfer ratios (MTRs) in eight patients with relapsing-remitting MS and in four control subjects were plotted as normalized histograms, and MTR parameters were compared with contrast-enhancing lesions and bulk white matter lesion load. RESULTS: In patients with relapsing-remitting MS, the histographic peak of 0.25+/-0.01 and the histographic mean of 0.21+/-0.01 were statistically lower than corresponding values in control subjects, in whom the histographic peak was 0.27+/-0.01 and the histographic mean was 0.23+/-0.01. When histograms (with MTRs ranging from 0.0 to 0.5) were analyzed by quartiles (quartile 1 to quartile 4) based on histographic area, voxels with low MTRs in quartile 1 (0 to 0.12) increased during the baseline period and corresponded to bulk white matter lesion load. Interferon beta-lb reduced enhancing lesions by 91% and mean bulk white matter lesion load by 15%, but had no effect on MTR in this patient cohort. CONCLUSION: Occult disease in normal-appearing white matter of patients with relapsing-remitting MS measured by MTR parallels the waxing and waning pattern of enhancing lesions and bulk white matter lesion load during the baseline period. MTR is not altered by interferon beta-lb, which raises the possibility of ongoing disease in normal-appearing white matter (not detected by conventional MR sequences).

Modulation of the expression of a multidrug resistance gene (mdr-1/P-glycoprotein) by differentiating agents.

Acquired resistance to multiple natural products in vitro is mediated by P-glycoprotein (Pgp). Expression of this protein has been demonstrated in some normal tissues and in tumor samples obtained from both untreated and treated patients. In situ hybridizations with RNA probes have demonstrated higher levels of expression of mdr-1/Pgp in well-differentiated tumors and in well-differentiated areas in tumors with mixed histologies. Expression of mdr-1/Pgp in human colon carcinoma cell lines was increased by the differentiating agents sodium butyrate, dimethyl sulfoxide, and dimethylformamide. In the SW-620 cell line addition of sodium butyrate resulted in a rapid induction of mdr-1/Pgp mRNA that was sustained for the duration of the exposure. The levels of P-glycoprotein were measured by immunoblotting and were also increased. Similar results were obtained in three other cell lines including the HCT-15 line. This induction occurred without alterations in nuclease sensitivity. Discontinuation of sodium butyrate was followed by a rapid fall in the levels of mRNA. The levels of P-glycoprotein returned to normal with a half-life of about 24 h. In spite of a 20-25-fold increase in the level of mdr-1/Pgp mRNA and P-glycoprotein, the SW-620 cell line did not demonstrate increased resistance to doxorubicin and vinblastine or decreased accumulation of vinblastine. In contrast, in the HCT-15 cell line, a 5-fold increase of mdr-1/Pgp was accompanied by a comparable fall in vinblastine accumulation which was reversed by verapamil. In the SW-620 cell line, the induced protein could be photolabeled using [3H]azidopine. Expression of mdr-1/Pgp appears to correlate with the degree of differentiation. However, its induction is not always accompanied by expression of the multidrug-resistance phenotype.

Stability and covalent modification of P-glycoprotein in multidrug-resistant KB cells.

An antipeptide antibody (P7) to P-glycoprotein has been produced by immunizing rabbits with a synthetic peptide. Antibody P7 is directed against the amino-terminal region of P170 (residues 28-35). The antibody immunoprecipitates a 170-kDa P-glycoprotein from extracts of drug-resistant KB-V1 cells that is not present in the drug-sensitive cell line KB-3-1. Antibody P7 was used to quantitate the amount of P-glycoprotein present in drug-resistant KB lines at various levels of resistance and to demonstrate the presence of P-glycoprotein in NIH 3T3 cells transfected with a cloned MDR1 cDNA or human genomic DNA encoding MDR1. Pulse-chase labeling experiments demonstrated that P-glycoprotein is synthesized as a 140-kDa precursor which is slowly converted over 2-4 h to a 170-kDa glycoprotein. Tunicamycin treatment blocks the conversion of the precursor to the mature form, and removal of N-linked oligosaccharides with Endo F reduces the relative molecular weight of P-glycoprotein to 140K. The mobility of mature P-glycoprotein is unaffected by treatment with neuraminidase and Endo H. These data indicate that P-glycoprotein is N-glycosylated and contains little or no neuraminic acid. P-Glycoprotein is also phosphorylated, and the extent of phosphate incorporated is proportional to the amount of protein present in drug-resistant cells.

Immunocytochemical localization of P170 at the plasma membrane of multidrug-resistant human cells.

P170 (P-glycoprotein) is a membrane protein found in high levels in multidrug-resistant cultured cell lines. We have localized this protein using monoclonal antibody MRK16 by immunofluorescence and electron microscopy in the multidrug-resistant human carcinoma cell line KB-C4. The P170 determinant recognized by antibody MRK16 was found on drug-resistant KB-C4 cells, but not on parental drug-sensitive KB-3-1 cells. The determinant was present on the external surface of the plasma membrane and on the luminal side of Golgi stack membranes. P170 was excluded from coated pits at the plasma membrane and absent from endocytic vesicles and lysosomes. This determinant was detected only in small amounts in the endoplasmic reticulum. The high protein concentration of P170 in the plasma membrane is consistent with a role of this protein as a drug efflux pump at the cell surface.

A novel fibrillar structure in cultured cells detected by a monoclonal antibody.

Using a monoclonal antibody, we have detected an antigen present in a unique fibrillar structure in the cytoplasm of cultured cells by immunofluorescence. These structures have been identified by transmission electron microscopy and ultrastructural immunocytochemistry as large single paracrystalline arrays of individual filaments morphologically similar to intermediate filaments. The antibody detects these structures in fibroblastic and epithelioid cultured cell lines of mouse, rat, bovine, and human origin but not of avian origin. Only a small percentage of the cells in a culture contains these structures; each cell usually contains only one, although two or more have been observed in a single cell. The structures are elongated vermiform arrays of filaments in the cytoplasm (approximately 0.5 X 3 microns) which have a thread-like or toroidal appearance. Because of this shape, we have named the putative antigen recognized by this antibody "nematin." Double-label experiments showed that these structures had no relationship to tubulin or vimentin. Immunocytochemical localization in human tissues revealed a high concentration of a reactive antigen in the stratum granulosum of skin and in what probably are neuroglial cells in the central nervous system. This monoclonal antibody may detect a novel intermediate filament protein and/or a shared determinant of different intermediate filament proteins.

Immunological similarity between the insulin receptor and the protein encoded by the src oncogene.

Insulin receptors resemble receptors for certain growth factors (epidermal growth factor, platelet-derived growth factor, and insulin-like growth factor I) in that all possess tyrosine-specific protein kinase activity. These cell surface receptors resemble protein kinases encoded by viral oncogenes in that both groups of enzymes phosphorylate proteins on tyrosine. Recently, we reported that there is immunological similarity between the insulin receptor and pp60src [the protein encoded by the src oncogene of Rous sarcoma virus (RSV)]. This is supported by the observation that anti-pp60src antiserum (TBR serum) immunoprecipitated radiolabeled insulin receptors derived from cultured human cells (IM-9 lymphoblasts and U-937 monocytes) and rabbit liver. Moreover, highly purified preparations of src protein inhibit the immunoprecipitation of insulin receptors by TBR serum, and the inhibition is correlated with the src kinase activity present in the preparation used. However, two observations suggested that there were immunological differences between pp60src and mammalian insulin receptors. 1) Even at a relatively high concentration (dilution, 1:10), TBR serum immunoprecipitated a relatively small percentage (approximately 20%) of the labeled insulin receptors. 2) Some lots of TBR serum with a high titer against pp60src failed to immunoprecipitate the insulin receptor. Viral oncogenes are thought to have been derived from proto-oncogenes in the host cell. Therefore, because the chicken is the natural host for RSV, we inquired whether there might be closer homology between pp60src and avian insulin receptors. Surprisingly, under conditions where TBR serum immunoprecipitates human insulin receptors, we could not detect immunoprecipitation of avian insulin receptors from chicken liver, chicken embryo fibroblasts, or turkey erythrocytes. The immunoprecipitation of human insulin receptor is not dependent on the method used for labeling the cells ([125I]insulin cross-linking), inasmuch as the receptor labeled by autophosphorylation with [gamma-32P]ATP could also be immunoprecipitated by TBR serum. These observations suggest that there is structural homology between pp60src and the insulin receptor (most likely the beta-subunit). Nevertheless, it seems unlikely that the insulin receptor gene is the proto-oncogene for the src gene of RSV.

Phorbol esters induce transient internalization without degradation of unoccupied epidermal growth factor receptors.

4 beta-Phorbol 12-myristate 13-acetate (PMA) treatment of KB cells at 37 degrees C rapidly induces a 50% reduction in epidermal growth factor (EGF) binding that is maximal by 30 min. EGF binding activity returns to the original value by 1 hr and remains constant for 2 hr thereafter. Using a polyclonal antibody directed against the cytoplasmic domain of the EGF receptor (EGF-R), we examined the fate of the receptor after PMA treatment. Immunofluorescent and electron microscopic localization of the EGF-R after PMA treatment demonstrated that about 50% of the receptor became internalized into endocytic vesicles (receptosomes) and Golgi-associated structures. Unlike EGF-induced internalization, PMA-induced internalization did not cause delivery of EGF-R to lysosomes or receptor degradation. Rather, receptor reappeared on the cell surface. No stimulation of EGF-R synthesis was observed after 1 hr of PMA treatment. Loss of cell surface binding correlated with the internalization of the EGF-R observed morphologically. A possible explanation for these observations is that PMA, an activator of protein kinase C, confers a signal sufficient for EGF-R clustering and internalization but not for transport to lysosomes.