Pharmacokinetics and Leukocyte Responses of Recombinant Human Interleukin-10

Purpose. To study the pharmacokinetics and ex vivo leukocyte responses of recombinant human IL-10 (rHuIL-10) following single SC and IV dosing. Methods. A randomized two-way cross-over study was undertaken in 17 healthy volunteers in which rHuIL-10 was administered as 25 g/ kg SC and IV doses. Blood samples were collected for 48 hr after dosing to determine serum IL-10 concentrations. Inhibitory activity of IL-10 on ex vivo production of inflammatory cytokines (TNF- and IL-1) by LPS-treated peripheral blood cells were measured over 96 hr. Results. A physiologically-relevant modeling approach was developed to determine the pharmacokinetics for two routes of administration (SC and IV). The IV dose showed polyexponential disposition with CL of 65 mL/kg/hr, V_ss of 70 mL/kg, and t1/2 of 1.94 hr. Absolute bioavailability averaged 42% for SC dosing which produced lower but sustained concentrations. Substantial and prolonged suppression of TNF- and IL-1 production was achieved during IL-10 treatment. The Hill Function was used to account for the joint concentration-dependent immunosuppressive action of rHuIL-10 after both IV and SC doses. The IC₅₀ values were about 0.03 ng/mL and I_max values were about 0.85 for both TNF- and IL-l suppression. The degree of change as well as the duration of leukocyte response was greater after SC administration than after IV administration. Conclusions. rHuIL-10 shows favorable PK/PD characteristics especially by theSC route of administration which produced prolonged suppression of cytokine production (ex vivo) which may be applicable in various immune-related disorders.     

Renal cyst pseudoenhancement: beam-hardening effects on CT numbers.

PURPOSE: To determine if simple renal cysts may be accurately characterized with helical computed tomography (CT) during peak levels of renal enhancement. MATERIALS AND METHODS: Water-filled "cysts" were suspended in varying concentrations of iodine solution, meant to simulate varying levels of renal enhancement, within an abdominal phantom. Volume-averaging effects were minimized by scanning cylindric 5-30-mm cysts with a helical technique (collimation, 5 mm; pitch, 1:1). Axial and helical techniques were then compared, and volume-averaging effects were evaluated by scanning 10- and 20-mm round cysts with 3-, 5-, and 7-mm collimation at background attenuation levels of 100 and 200 HU. RESULTS: Cylindric cyst attenuation increased consistently with increasing background attenuation. As background attenuation increased by 90 HU, attenuation increased by 11-17 HU in small (5- or 10-mm) cysts, and by 7-9 HU in large (15-30-mm) cysts. As background attenuation increased by 180 HU, attenuation increased by 18-28 HU in small cysts and by 10-15 HU in large cysts. Spherical cyst attenuation differences were maximized when smaller cysts were imaged with larger collimation, which is when volume-averaging effects became apparent. Axial and helical CT numbers did not differ substantially. Computer simulation studies showed that the observed effect could not be explained by beam hardening alone. CONCLUSION: Pseudoenhancement of renal cysts may occur if helical CT is performed during peak renal enhancement. CT algorithm modification may be necessary to correct for this effect, which is likely related to an inadequate algorithmic correction for beam hardening.     

Listeriolysin generates a route for the presentation of exogenous antigens by major histocompatibility complex class I

We have exploited the pore forming activity of listeriolysin, the hemolysin of Listeria monocytogenes, to activate CD8⁺ T cells with soluble proteins in vivo and in vitro. Immunization with soluble, hemolytically active listeriolysin induces both cytotoxic CD8⁺ T cells and CD4⁺ T cells, and the CD8⁺ T cells can be propagated with soluble listeriolysin in vitro. Moreover, conventional antigens like ovalbumin mixed together with listeriolysin are also efficiently introduced into the MHC class I pathway in vitro and in vivo. Hence, listeriolysin effectively directs itself and passenger molecules into the intracellular compartment that leads to the cytotoxic T cell response. In this way, we circumvent the bias of CD8⁺ T cells to recognize intracellular antigens presented by major histocompatibility complex class I molecules. As cytotoxic CD8⁺ T cells are of pivotal importance in eliminating viral and microbial pathogens, the findings reported here could prove to be useful in vaccine development.     

Differential deletions in 3p are associated with the development of head and neck squamous cell carcinoma in Indian patients

In this study we performed detailed deletion mapping of two broad regions in the short arm (p) of chromosome 3 (i.e., 3p21.2 approximately p22 and 3p12 approximately p13), which were shown to have a high rate of deletions in head and neck lesions in our previous study. Using 18 highly polymorphic microsatellite markers, the deletion mapping was done in 35 dysplastic lesions and 46 primary head and neck squamous cell carcinoma (HNSCC) samples from Indian patients. Within the 21.6-megabase (Mb) region of 3p21.1 approximately p21.33, we have identified four areas (D1, 3p21.33; D2, 3p21.32; D3, 3p21.31; D4, 3p21.1) that showed a high frequency (46%-69%) of deletions in our samples. In the 3p12 approximately p13 region, we narrowed down the deletion within the 0.7-Mb region (D5, 3p12.1). Among these five regions (D1-D5), deletion in D3 is suggested to be necessary for the development of early dysplastic lesions, whereas the deletion in D2 may be necessary for dysplastic lesions and tumor progression. On the other hand, the deletion in D5 is significantly associated with progression of the lesions from mild/moderate to severe dysplasia. The deletions in D1 and D4, however, are required for tumor progression. As in our previous study, microsatellite size alterations (MA) were observed to be high in and around the highly deleted regions and gradually increased during the progression of the tumor. Loss of normal copy/interstitial alterations of chromosome 3 in the late stages of the tumor as well as rare biallelic alterations around the highly deleted regions also were seen in our samples. Human papilloma virus infection has been found to be associated with the deletion in the D5 region and MA in the D1 region, whereas nodal involvement of the tumor correlated only with the MA in D1 and D5. Thus, this study indicates that multiple tumor suppressor genes whose differential deletions are associated with the development of HNSCC may be present in 3p.     

Melanoma × macrophage hybrids with enhanced metastatic potential

Studies were conducted on the hypothesis that melanoma metastasis might be initiated through the gener-ation of hybrids comprised of cells of the primary tumor and tumor-infiltrating leukocytes. Fusion hybrids were generated in vitro between weakly metastatic Cloudman S91 mouse melanoma cells and normal mouse or human macrophages. Hybrids were implanted s.c. in the tail and mice were monitored for metastases. Controls included parental S91 cells, autologous S91 × S91 hybrids, and B16F10 melanoma cells. Of 35 hybrids tested, most were more aggressive than the parental melanoma cells, producing metastases sooner and in more mice. A striking characteristic was heterogeneity amongst hybrids, with some lines producing no metastases and others producing metastases in up to 80% of mice. With few exceptions, hybrids with the highest metastatic potential also had the highest basal melanin content whereas those with the lowest metastatic potential were basally amelanotic, as were t he parental melanoma cells. A spontaneous in vivo supermelanotic hybrid between an S91 tumor cell and DBA/2J host cell was one of the most metastatic lines. Hybrids with the highest metastatic potential also exhibited markedly higher chemotaxis to fibroblast-conditioned media. Histologically, the metastatic hybrids demonstrated vascular invasion and spread to distant organs similar to that of metastatic melanomas in mice and humans. Thus previous findings of enhanced metastasis in leukocyte × lymphoma hybrids can now be extended to include leukocyte × melanoma hybrids. Whether such hybridization is a natural cause of metastasis in vivo remains to be determined; however the fusion hybrids with genetically-matched parents described herein so closely resembled naturally- occurring metastatic melanoma cells that they could serve as useful new models for studies of this complex and deadly phenomenon. © Rapid Science Ltd.     

Interaction of Listeria monocytogenes with mouse dendritic cells.

In this study, the interaction of murine dendritic cells with Listeria monocytogenes was investigated. Dendritic cells are efficient antigen-presenting cells, play a key role in the immune response, and are capable of migrating over substantial distances between sites of infection and lymphoid tissues. L. monocytogenes EGD invaded dendritic cells, escaped from phagosomes into the cytoplasm, and there directed actin nucleation, polymerization, and polarization in a typical fashion, thereby achieving intracellular movement and cell-to-cell spread. The internalization process appears to be independent of the inl locus. Interestingly, an intact microtubular function was essential for efficient uptake, whereas in a previous report, microtubule disruption did not affect bacterial spread in Caco-2 cells. The results obtained also suggest that L. monocytogenes binds to glycosylated receptors of dendritic cells. Uptake of Listeria cells was mediated by a protein kinase-dependent transducing phosphorylation signal that induces the actin polymerization-dependent phagocytic process. To achieve efficient uptake, de novo protein synthesis of eukaryotic and prokaryotic cells is also required. Despite the killing of dendritic cells, wild-type bacteria were found to persist in small numbers in some cells for at least 24 h. When different isogenic mutants of the EGD strain were analyzed for their capability to interact with dendritic cells, it was observed that some virulence-attenuated mutants (i.e., prfA and delta hly) persisted in large numbers for even longer times. Invasion of dendritic cells by L. monocytogenes, which in turn could result in either cell death or persistent infection, might have an important role in the pathogenesis of listeriosis, leading to impaired immune responses with inefficient bacterial clearance and/or promoting bacterial spread.     

CD8+ lineage dendritic cells determine adaptive immune responses to inflammasome activation upon sterile skin injury

The molecular links between sterile inflammation and induction of adaptive immunity have not been fully identified. Here, we examine how damage‐associated molecular patterns (DAMPs), as opposed to pathogen‐associated molecules (PAMPs), regulate the immune response to non–self‐antigens presented at the site of a physical injury. Heat applied briefly to the skin invokes sterile inflammation, characterized by local cell death and caspase‐1 activation without demonstrably disrupting skin integrity. Co‐delivery of ovalbumin (OVA) with heat injury induces OVA‐specific CD8⁺ T‐cell responses, and this is dependent on caspase‐1 activation and MyD88 signalling. Using Id2flox/flox‐CD11cCre+ mice, we demonstrate that CD8⁺ lineage DCs are required to induce OVA‐specific CD8⁺ T‐cell responses following heat injury. Consistent with this observation, intradermal administration of CD8⁺ lineage DCs but not CD11b⁺ lineage DCs restores priming of CD8⁺ T‐cell responses in Casp‐1^?/? mice. Thus, we conclude that a sterile injury induces CD8⁺ T‐cell immune responses to local antigen through caspase‐1 activation and requires CD8⁺ lineage DCs, a finding of significance for immunotherapy and for the pathogenesis of autoimmunity.