Robert I. Tepper

Identification and Expression Cloning of a Leptin Receptor, OB-R

The ob gene product, leptin, is an important circulating signal for the regulation of body weight. To identify high affinity leptin-binding sites, we generated a series of leptin-alkaline phosphatase (AP) fusion proteins as well as [125I]leptin. After a binding survey of cell lines and tissues, we identified leptin-binding sites in the mouse choroid plexus. A cDNA expression library was prepared from mouse choroid plexus and screened with a leptin-AP fusion protein to identify a leptin receptor (OB-R). OB-R is a single membrane-spanning receptor most related to the gp130 signal-transducing component of the IL-6 receptor, the G-CSF receptor, and the LIF receptor. OB-R mRNA is expressed not only in choroid plexus, but also in several other tissues, including hypothalamus. Genetic mapping of the gene encoding OB-R shows that it is within the 5.1 cM interval of mouse chromosome 4 that contains the db locus.

Evidence That the Diabetes Gene Encodes the Leptin Receptor: Identification of a Mutation in the Leptin Receptor Gene in db/db Mice

OB-R is a high affinity receptor for leptin, an important circulating signal for the regulation of body weight. We identified an alternatively spliced transcript that encodes a form of mouse OB-R with a long intracellular domain. db/db mice also produce this alternatively spliced transcript, but with a 106 nt insertion that prematurely terminates the intracellular domain. We further identified G --> T point mutation in the genomic OB-R sequence in db/db mice. This mutation generates a donor splice site that converts the 106 nt region to a novel exon retained in the OB-R transcript. We predict that the long intracellular domain form of OB-R is crucial for initiating intracellular signal transduction, and as a corollary, the inability to produce this form of OB-R leads to the severe obese phenotype found in db/db mice.

Murine interleukin-4 displays potent anti-tumor activity in vivo

We have devised a sensitive means to assess the anti-tumor effect of cytokines that act via the mobilization of host-mediated defenses. This assay involves transfecting malignant cells to produce a specific cytokine (in this case, IL-4) and measuring the ability of transfectants to form tumors alone and when mixed with a variety of nontransfected tumor cells. In this way, we have identified a potent, non-cell autonomous, anti-tumor effect of IL-4 which is effective against a wide range of tumor cell types in vivo. The effect is reversed by anti-IL-4 antibody, correlates closely with levels of IL-4 production, and is evident in nu/nu mice. The anti-tumor effect seems to be mediated by an inflammatory infiltrate composed of eosinophils and macrophages.

IL-4 induces allergic-like inflammatory disease and alters T cell development in transgenic mice

We have assessed the biologic role of IL-4 by fusing its gene to an immunoglobulin promoter/enhancer and introducing it into transgenic mice. By attenuating the transgene promoter through the insertion of E. coli lac operator sequences, we have created a series of animals that constitutively express varying amounts of IL-4. Overexpression of IL-4 results in a marked increase in serum IgE levels and the appearance of an inflammatory ocular lesion (blepharitis) with characteristic histopathologic features seen in allergic reactions. In addition, expression of the IL-4 transgene in the thymus perturbs T cell maturation, reducing the population of immature CD4+CD8+ thymocytes and peripheral T cells while increasing the population of mature CD8+ thymocytes. These results demonstrate that deregulation of a single cytokine gene in vivo can induce a complex inflammatory reaction resembling that observed in human allergic disease.

Analysis of K-ras oncogene mutations in chronic pancreatitis with ductal hyperplasia☆

BACKGROUND K-ras oncogene mutations have been identified in up to 95% of pancreatic cancers, implying their critical role in their molecular pathogenesis. However, the earliest stage in which K-ras mutations can be detected in potential precursor lesions of pancreatic cancer remains unclear. This study evaluates pancreatic ductal hyperplasia in the setting of chronic pancreatitis, which predisposes to pancreatic cancer development, for K-ras codon 12 and 13 mutations. METHODS Paraffin-embedded surgical specimens from 42 patients with chronic pancreatitis were examined microscopically for the presence of ductal hyperplasia. Both hyperplastic and nonhyperplastic ducts were microdissected from the specimens that contained hyperplasia (11 of 42). Four of the remaining specimens without hyperplasia served as controls. Genomic DNA was extracted, and polymerase chain reaction and amplification of the K-ras oncogene was performed. Polymerase chain reaction products were evaluated by means of hybridization to mutant specific oligonucleotide probes and by means of automated DNA sequencing. RESULTS K-ras codon 12 mutations representing glycine to valine substitutions were present in 2 of (18%) 11 patients with ductal hyperplasia. No mutations were found in the controls without ductal hyperplasia. CONCLUSIONS Our study supports the premise that K-ras mutations develop in a subset of chronic pancreatitis associated hyperplasia and provides a genetic basis for the potential progression of chronic pancreatitis to pancreatic cancer.

An implantable microdevice to perform high-throughput in vivo drug sensitivity testing in tumors

An implantable microdevice is demonstrated to release microdoses of multiple drugs into confined regions of tumors and allows for assessment of each drug’s efficacy to identify optimal therapy. Drug-releasing implant tests cancer’s resolve Predicting whether a patient will respond to a drug is not easy, often relying on empirical evidence. Toward personalized medicine, animal models of patient tumors have been developed as well as engineered cell-material combinations meant to replicate a tumor in vitro. But the true test of whether a tumor responds to a drug will be by evaluating the tumor itself, within its own microenvironment. To this end, Jonas et al. created miniature drug delivery vessels that can be implanted with a standard biopsy needle directly into the tumor. These vessels, less than 1 mm in diameter, contained up to 16 microwells that each released a bolus of drug into the surrounding tumor tissue. The device and its surrounding tissue were then removed with a larger coring needle to see if the cancer cells had responded to the drug—or combination of drugs. In mouse models of melanoma, breast, or prostate cancers, the local response to a common chemotherapeutic, doxorubicin, matched the tumor response to systemic therapy. Furthermore, in a mouse model of triple-negative breast cancer, tumor sensitivity to five different locally delivered cancer drugs was identical to tumor response after intravenous administration of drug; for instance, tumors were most responsive to paclitaxel and least responsive to lapatinib. Such tiny drug-releasing devices can be implanted at different locations within the tumor, overcoming issues with tumor heterogeneity, and allowing for reproducible evaluation of drug sensitivity directly within the patient. Current anticancer chemotherapy relies on a limited set of in vitro or indirect prognostic markers of tumor response to available drugs. A more accurate analysis of drug sensitivity would involve studying tumor response in vivo. To this end, we have developed an implantable device that can perform drug sensitivity testing of several anticancer agents simultaneously inside the living tumor. The device contained reservoirs that released microdoses of single agents or drug combinations into spatially distinct regions of the tumor. The local drug concentrations were chosen to be representative of concentrations achieved during systemic treatment. Local efficacy and drug concentration profiles were evaluated for each drug or drug combination on the device, and the local efficacy was confirmed to be a predictor of systemic efficacy in vivo for multiple drugs and tumor models. Currently, up to 16 individual drugs or combinations can be assessed independently, without systemic drug exposure, through minimally invasive biopsy of a small region of a single tumor. This assay takes into consideration physiologic effects that contribute to drug response by allowing drugs to interact with the living tumor in its native microenvironment. Because these effects are crucial to predicting drug response, we envision that these devices will help identify optimal drug therapy before systemic treatment is initiated and could improve drug response prediction beyond the biomarkers and in vitro and ex vivo studies used today. These devices may also be used in clinical drug development to safely gather efficacy data on new compounds before pharmacological optimization.

Thumb replantation: a retrospective review of 103 cases.

Replantation has become the state of the art reconstruction for an amputated thumb. The aim of our study was to review our series of thumb replantations over a period of 12 years at the Bellevue Hospital Center in New York City. The mechanism of injury, level of amputation, and use of vein grafts was reviewed and correlated with survival rates of the replanted thumbs. The overall survival rate was 91.3%. Of the 12 thumbs that were re-explored for vascular compromise, 75% were successfully salvaged. Our study also indicates that there is no statistical difference in survival of thumb replants when comparing the mechanism of injury, the level of amputation, and the use of vein grafts. However, the use of vein grafting seemed to be beneficial in the successful outcome of replantation in severe crush and avulsion injuries, even though the values did not reach statistical significance. We conclude that thumb replantation is associated with very high survival rate, regardless of the mechanism of injury or level of amputation, and should be attempted in all cases. An early reexploration for vascular problems yields a high salvage rate and should be performed in all cases. We also recommend the use of vein grafts in severe crush and avulsion injuries.

Analysis of Mast Cell Activation Using Diamine Oxidase-Gold Enzyme-Affinity Ultrastructural Cytochemistry

We review a new technique--diamine oxidase (DAO)-gold ultrastructural enzyme-affinity labeling--which we developed to localize histamine in subcellular sites of mast cells. The DAO-gold method showed that isolated human lung mast cells contained abundant histamine in their cytoplasmic granules, a conclusion which was verified by a large number of specificity controls. We also studied mast-cell-rich eyelid lesions which developed in interleukin-4 transgenic mice. The DAO-gold method demonstrated histamine in the electron-dense granules of mast cells in these lesions, but little or no histamine was detected in the swollen, empty granules of mast cells undergoing piecemeal degranulation. This new enzyme-affinity-gold method has permitted the first ultrastructural localization of histamine in subcellular sites of routinely prepared electron microscopy samples. The method has also permitted the first morphological studies of histamine secretion in vivo and has demonstrated that such secretion can be associated with the ultrastructural changes of piecemeal degranulation.

Novel Strategies for Cancer Treatment through Modulation of Proimmune and Proinflammatory Cytokines

The remarkable progress in understanding the molecular basis of the immune system has prompted new strategies for the treatment of experimental and human cancers. In particular, the cloning and identification of the biological activity of cytokine genes has allowed for the activation and expansion of various hematolymphoid populations with cytotoxic potential. Extrapolation from results obtained with cytokines in biological systems in vitro and in vivo suggest that, in general, cytokines act potently to modulate immune and inflammatory responses locally, but rarely mediate systemic activation of immune and inflammatory cells. The localized action of cytokines is also predictable from their release by helper T lymphocyte (and other cell) populations upon exposure to an antigenic challenge which, in most physiological situations, is a localized event. In terms of activating cytotoxic cells for the killing of tumors, this property of cytokine action must be considered. This report describes strategies that have been developed to direct T cell dependent and independent cytotoxicity against established cancers in experimental animals. These strategies have relied upon the identification of specific proinflammatory and proimmune properties of various cytokines with respect to tumor cells. The use of viral vectors enabling the targeting of cytokine gene products to sites of tumor cell growth have led to gene therapy strategies which take advantage of the potent localized actions of these proteins.