Citations

Previously, using a synthetic peptide strategy, we determined that the region of the common glycoprotein hormone alpha subunit between residues 26 and 46 is a site of interaction of the hormone with the thyroid membrane-bound receptor for thyroid-stimulating hormone (TSH). We have undertaken to identify further the specific residues within this 21-amino acid span that are critical in hormone receptor binding. We synthesized three nested sets of peptide, two in which we systematically truncated the amino-terminal region of the sequence and another in which we truncated the carboxyl-terminal region, and we synthesized a fourth nested set in which we systematically substituted alanine for the native residues from the region of highest activity. Each peptide was tested in a TSH radioreceptor assay for its ability to inhibit binding of 125I-labeled bovine TSH to porcine thyroid membranes. Removal either by truncation or alanine substitution, of several specific residues resulted in a significant reduction in the ability of the sequence to interact with receptor; these residues included Cys31, Cys32, Phe33, Arg35, Arg42, Lys44, and Lys45, suggesting that they are crucial for binding activity. Loss of activity also occurred with substitution for Gly30 and Ser34, but the reduction was less pronounced. Amino-terminal truncation of the sequence through Arg35 (leaving the alpha-subunit peptide 36-46) resulted in greater than 98% loss of activity of the sequence. We conclude that two distinct receptor binding regions lie within the alpha-subunit 26-46 sequence. The first lies between residues Gly30 and Arg35 and includes Cys31, Cys32, and Phe33 as important constituents, and the second region lies between residues Arg42 and Lys45 and includes Lys44 as an important residue and Ser43 as a less important component.

Binding of human [ 125 I]luteinizing hormone to homogenates of luteinized rat ovaries is dependent upon time, temperature, and pH and is saturable. Injection of human chorionic gonadotropin in vivo or addition of unlabeled human chorionic gonadotropin or human or ovine luteinizing hormone in vitro inhibits binding, whereas follicle stimulating hormone or prolactin is without effect. The dissociation constant for binding of luteinizing hormone receptor is 0.79 nM. The number of binding sites is 94 femtomol/mg of wet weight. NaCl, KCl, MgSO 4 , and CaCl 2 at concentrations below 10 mM have no effect on binding, while all salts significantly inhibit binding at 150 mM. Binding of [ 125 I]luteinizing hormone to its receptors is destroyed by proteolytic enzymes and by phospholipase C and D. The total binding activity is quantitatively recovered in the 2000 × g pellet of the homogenate.

AbstractMetastatic renal cell carcinoma (RCC) is an aggressive disease refractory to most existing therapeutic modalities. Identifying new markers for disease progression and drug targets for RCC will benefit this unmet medical need. We report a subset of clear cell and papillary cell RCC aberrantly expressing the lymphocyte activation marker CD70, a member of the tumor necrosis factor superfamily. Importantly, CD70 expression was found to be maintained at the metastatic sites of RCC. Anti-CD70 antibody-drug conjugates (ADC) consisting of auristatin phenylalanine phenylenediamine (AFP) or monomethyl auristatin phenylalanine (MMAF), two novel derivatives of the anti-tubulin agent auristatin, mediated potent antigen-dependent cytotoxicity in CD70-expressing RCC cells. Cytotoxic activity of these anti-CD70 ADCs was associated with their internalization and subcellular trafficking through the endosomal-lysosomal pathway, disruption of cellular microtubule network, and G2-M phase cell cycle arrest. The efficiency of drug delivery using anti-CD70 as vehicle was illustrated by the much enhanced cytotoxicity of antibody-conjugated MMAF compared with free MMAF. Hence, ADCs targeted to CD70 can selectively recognize RCC, internalize, and reach the appropriate subcellular compartment(s) for drug release and tumor cell killing. In vitro cytotoxicity of these ADCs was confirmed in xenograft models using RCC cell lines. Our findings provide evidence that CD70 is an attractive target for antibody-based therapeutics against metastatic RCC and suggest that anti-CD70 ADCs can provide a new treatment approach for advanced RCC patients who currently have no chemotherapeutic options. (Cancer Res 2006; 66(4): 2328-37)

A transgenic mouse containing the complete human SLAM (hSLAM/CD150) gene, including its endogenous promoter for transcription, was generated by using human genomic DNA cloned into a bacterial artificial chromosome. hSLAM, the primary receptor for measles viruses (MV), was expressed on activated B, T, and dendritic cells with an expression profile equivalent to that of humans. We demonstrated that hSLAM + cells obtained from the transgenic mouse, including activated B, T, and dendritic cells, were susceptible to MV infection in a receptor-dependent manner. Evidence was provided for transient infection in the nasal lymph nodes of hSLAM + mice after intranasal inoculation. Virus was rapidly cleared without signs of secondary replication. To improve the efficiency of MV production, the hSLAM + mice were bred with mice having a Stat1 -deficient background. These mice were more susceptible to MV infection and produced more virus particles. After intranasal and intraperitoneal inoculation of these mice with MV, infections of the thymus, spleen, nasal, mesenteric, and leg lymph nodes were detected. Upon necropsy, enlarged lymph nodes and spleen were apparent. Flow cytometric analysis showed that abnormally large numbers of mature neutrophils and natural killer cells caused the splenomegaly. The hSLAM transgenic mouse constitutes an improved rodent model for studying the interaction of MV with immune cells that more accurately reflects the infection pattern found in humans.

SummaryImmunity to measles is conferred by the interplay of humoral and cellular immune responses, the latter being critical in maintaining long-term recall response. Therefore, it is important to evaluate measles-specific humoral and cellular immunity in populations several years after vaccination and understand the correlations among these measures of immunity. We examined measles-specific antibodies, lymphoproliferation and the Th1/Th2 signature cytokines, interferon (IFN)-γ and interleukin (IL)-4, in a population-based cohort of healthy children from Olmsted County, Minnesota after two doses of measles–mumps–rubella-II (MMR-II) vaccine. We detected positive measures of measles-specific cellular and humoral immunity in the majority of our study population. However, a small proportion of subjects demonstrated an immune response skewed towards the Th2 type, characterized by the presence of either IL-4 and/or measles-specific antibodies and a lack of IFN-γ production. Further, we observed a significant positive correlation between lymphoproliferation and secretion of IFN-γ (r = 0·20, P = 0·0002) and IL-4 (r = 0·15, P = 0·005). Measles antibody levels were correlated with lymphoproliferation (r = 0·12, P = 0·03), but lacked correlation to either cytokine type. In conclusion, we demonstrated the presence of both long-term cellular and humoral responses after MMR-II vaccination in a significant proportion of study subjects. Further, a positive correlation between lymphoproliferation and IL-4 and IFN-γ suggests that immunity to measles may be maintained by both Th1 and Th2 cells. We speculate that the Th2 biased response observed in a subset of our subjects may be insufficient to provide long-term immunity against measles. Further examination of the determinants of Th1 versus Th2 skewing of the immune response and long-term follow-up is needed.

AbstractThe latest World Health Organization International Classification defines papillary thyroid carcinoma by its “follicular cell differentiation. .. as well as characteristic nuclear changes”. However the oxyphilic (Hürthle cell) papillary carcinoma have nuclei which generally resemble the nuclei seen in oxyphilic follicular carcinomas, and such oxyphilic papillary tumors may behave more aggressively than typical papillary cancers. To further characterize these rare tumors, we identified during a 32‐year period 22 patients with oxyphilic papillary cancer and compared them with 1,084 patients with typical papillary cancers and 57 patients with oxyphilic follicular cancers treated by the Mayo surgical group during the same time period. Although typical papillary and oxyphilic papillary cancers were comparable with regards to patient age, tumor size and extent, TNM stage, and prognostic score (AGES), there were significant differences. Compared to typical papillary tumors, oxyphilic papillary cancers had fewer neck nodal metastases at primary diagnosis (5% vs 40%,p<0.0001), were more often DNA non‐diploid (71% vs 21%,p<0.001), and after 10 postoperative years had higher rates of both tumor recurrence (28% vs 11%,p<0.0001) and cause‐specific mortality (1.7% vs 4%,p<0.0005). In these four important respects the oxyphilic papillary cancers more resembled the oxyphilic follicular cancers. For oxyphilic follicular cancers, the frequency of initial neck nodal metastases was 7% (cf 5%); 83% of the oxyphilic follicular tumors were non‐diploid (cf 71%), and at 10 years postoperatively the tumor recurrence and cause‐specific mortality rates were 28% and 18%, insignificantly different from 28% and 17% seen with the oxyphilic papillary cancers. These results demonstrate that oxyphilic papillary tumors are more similar to oxyphilic follicular than typical papillary cancers, and suggest that in a differentiated follicular cell‐derived carcinoma a predominance of oxyphilic cells may be a prognostically more relevant feature than the individual tumor's predominant papillary or follicular morphologic pattern. Perhaps in future Histologic Classifications the World Health Organization should group the oxyphilic papillary cancers with the oxyphilic follicular rather than the typical papillary carcinomas.

AbstractDistinguishing cellular follicular adenomas (FA) from minimally invasive follicular carcinomas (FC) continues to plague even experienced thyroid pathologists. DNA ploidy analysis has been promoted as a means of making this differentiation; however, the finding of DNA aneuploidy in FA has caused concern that they may demonstrate potential for malignant behavior or should even be reclassified as low‐grade noninvasive cancers. In histologically‐proven FC, nuclear DNA content has been claimed to have predictive power equivalent to that of all other prognostic factors combined. The aims of the present study, therefore, were to define the DNA ploidy characteristics of FA and FC, to assess the diagnostic potential of cell‐cycle parameters, and, in FC, to investigate the prognostic role of such measurements.We measured DNA content of 124 tumors (60 FA, 64 FC). DNA pattern was normal (diploid) in 75% of FA and 45% of FC, tetraploid/polyploid (T/P) in 13% of FA and 25% of FC, and aneuploid in 12% of FA and 30% of FC. FC was histologically verified in 39% of DNA normal, 67% of T/P, and 73% of aneuploid tumors. DNA index, S‐phase, G2M, and S‐phase plus G2M were analyzed and were not helpful in differentiating between FA and FC. No patient with FA developed tumor recurrence. In FC (excluding the Hürthle cell variant), no significant differences were found among the 3 DNA ploidy groups with respect to either cancer death or tumor recurrence; however, combining the Hürthle cell variant of follicular carcinomas with pure follicular carcinomas, the presence of distant metastases, DNA aneuploidy, and patient age were the only independently significant prognostic variables. Based on these data, while not valuable for determining the presence of malignancy, DNA ploidy determination is of major prognostic value in follicular carcinomas (including the Hürthle cell variant).