Intraperitoneal immunotherapy: historical perspectives and modern therapy.

Intraperitoneal immunotherapy represents a novel strategy for the management of peritoneal metastases (PM). Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) has remained the gold standard of treatment for patients with PM, yet despite optimal treatment, recurrence rates remain high and long-term survival poor. From Coley's toxins to immune checkpoint inhibitors, the wide variety of anticancer immunotherapeutic strategies are now garnering attention for control of regional disease of the peritoneal cavity. Early studies with vaccine-based therapies, adoptive cell transfer, immune checkpoint inhibitors, and chimeric T cells with tumor-specific antigen receptors (CAR-T cells) are being performed, showing promise for control of peritoneal spread and induction of lasting anticancer immunity. In addition, catumaxomab, a trifunctional antibody, has been approved for intraperitoneal immunotherapy in Europe for the control of malignant ascites in patients with epithelial cell adhesion molecule positive cancers. We review a brief history of immunotherapy and current modalities under investigation for intraperitoneal use in the treatment of PM.

The dose distribution of low dose rate Cs-137 in intracavitary brachytherapy: comparison of Monte Carlo simulation, treatment planning calculation and polymer gel measurement.

In this study, the dose distribution delivered by low dose rate Cs-137 brachytherapy sources was investigated using Monte Carlo (MC) techniques and polymer gel dosimetry. The results obtained were compared with a commercial treatment planning system (TPS). The 20 mm and the 30 mm diameter Selectron vaginal applicator set (Nucletron) were used for this study. A homogeneous and a heterogeneous-with an air cavity-polymer gel phantom was used to measure the dose distribution from these sources. The same geometrical set-up was used for the MC calculations. Beyond the applicator tip, differences in dose as large as 20% were found between the MC and TPS. This is attributed to the presence of stainless steel in the applicator and source set, which are not considered by the TPS calculations. Beyond the air cavity, differences in dose of around 5% were noted, due to the TPS assuming a homogeneous water medium. The polymer gel results were in good agreement with the MC calculations for all the cases investigated.

Polymer gel measurement of dose homogeneity in the breast: comparing MLC intensity modulation with standard wedged delivery.

Polymer gel dosimetry has been used to measure the radiotherapy dose homogeneity in a breast phantom for two different treatment methods. The first 'standard' method uses two tangential wedged fields while the second method has three static fields shaped by multileaf collimators (MLCs) in addition to the standard wedged fields to create intensity modulated fields. Gel dose distributions from the multileaf modulation treatment show an improved dose uniformity in comparison to the standard treatment with a decreased volume receiving doses over 105%.

Three-dimensional hydrodynamic lattice-gas simulations of binary immiscible and ternary amphiphilic flow through porous media.

We report the results of a study of multiphase flow in porous media. A Darcy's law for steady multiphase flow was investigated for both binary and ternary amphiphilic flow. Linear flux-forcing relationships satisfying Onsager reciprocity were shown to be a good approximation of the simulation data. The dependence of the relative permeability coefficients on water saturation was investigated and showed good qualitative agreement with experimental data. Nonsteady-state invasion flows were investigated, with particular interest in the asymptotic residual oil saturation. The addition of surfactant to the invasive fluid was shown to significantly reduce the residual oil saturation.

Three-dimensional hydrodynamic lattice-gas simulations of domain growth and self-assembly in binary immiscible and ternary amphiphilic fluids.

We simulate the dynamics of phase assembly in binary immiscible fluids and ternary microemulsions using a three-dimensional hydrodynamic lattice-gas approach. For critical spinodal decomposition we perform the scaling analysis in reduced variables introduced by Jury et al. [Phys. Rev. E 59, R2535 (1999)] and by Bladon et al. [Phys. Rev. Lett. 83, 579 (1999)]. We find a late-stage scaling exponent consistent with the R approximately t(2/3) inertial regime. However, as observed with the previous lattice-gas model of Appert et al. [J. Stat. Phys. 81, 181 (1995)] our data do not fall in the same range of reduced length and time as those of Jury et al. and Bladon et al. For off-critical binary spinodal decomposition we observe a reduction of the effective exponent with decreasing volume fraction of the minority phase. However, the n=1 / 3 Lifshitz-Slyzov-Wagner droplet coalescence exponent is not observed. Adding a sufficient number of surfactant particles to a critical quench of binary immiscible fluids produces a ternary bicontinuous microemulsion. We observe a change in scaling behavior from algebraic to logarithmic growth for amphiphilic fluids in which the domain growth is not arrested. For formation of a microemulsion where the domain growth is halted we find that a stretched exponential growth law provides the best fit to the data.

Targeted deletion of the gene encoding iron regulatory protein-2 causes misregulation of iron metabolism and neurodegenerative disease in mice.

In mammalian cells, regulation of the expression of proteins involved in iron metabolism is achieved through interactions of iron-sensing proteins known as iron regulatory proteins (IRPs), with transcripts that contain RNA stem-loop structures referred to as iron responsive elements (IREs). Two distinct but highly homologous proteins, IRP1 and IRP2, bind IREs with high affinity when cells are depleted of iron, inhibiting translation of some transcripts, such as ferritin, or turnover of others, such as the transferrin receptor (TFRC). IRPs sense cytosolic iron levels and modify expression of proteins involved in iron uptake, export and sequestration according to the needs of individual cells. Here we generate mice with a targeted disruption of the gene encoding Irp2 (Ireb2). These mutant mice misregulate iron metabolism in the intestinal mucosa and the central nervous system. In adulthood, Ireb2(-/-) mice develop a movement disorder characterized by ataxia, bradykinesia and tremor. Significant accumulations of iron in white matter tracts and nuclei throughout the brain precede the onset of neurodegeneration and movement disorder symptoms by many months. Ferric iron accumulates in the cytosol of neurons and oligodendrocytes in distinctive regions of the brain. Abnormal accumulations of ferritin colocalize with iron accumulations in populations of neurons that degenerate, and iron-laden oligodendrocytes accumulate ubiquitin-positive inclusions. Thus, misregulation of iron metabolism leads to neurodegenerative disease in Ireb2(-/-) mice and may contribute to the pathogenesis of comparable human neurodegenerative diseases.

Combined natural killer cell and dendritic cell functional deficiency in KARAP/DAP12 loss-of-function mutant mice.

KARAP/DAP12 is a transmembrane polypeptide with an intracytoplasmic immunoreceptor tyrosine-based activation motif (ITAM). KARAP/DAP12 is associated with several activating cell surface receptors in hematopoietic cells. Here, we report that knockin mice bearing a nonfunctional KARAP/DAP12 ITAM present altered innate immune responses. Although in these mice NK cells are present and their repertoire of inhibitory MHC class I receptors is intact, the NK cell spectrum of natural cytotoxicity toward tumor cell targets is restricted. KARAP/DAP12 loss-of-function mutant mice also exhibit a dramatic accumulation of dendritic cells in muco-cutaneous epithelia, associated with an impaired hapten-specific contact sensitivity. Thus, despite its homology with CD3zeta and FcRgamma, KARAP/DAP12 plays a specific role in innate immunity, emphasizing the nonredundancy of these ITAM-bearing polypeptides in hematopoietic cells.

RIBP, a novel Rlk/Txk- and itk-binding adaptor protein that regulates T cell activation.

A novel T cell-specific adaptor protein, RIBP, was identified based on its ability to bind Rlk/Txk in a yeast two-hybrid screen of a mouse T cell lymphoma library. RIBP was also found to interact with a related member of the Tec family of tyrosine kinases, Itk. Expression of RIBP is restricted to T and natural killer cells and is upregulated substantially after T cell activation. RIBP-disrupted knockout mice displayed apparently normal T cell development. However, proliferation of RIBP-deficient T cells in response to T cell receptor (TCR)-mediated activation was significantly impaired. Furthermore, these activated T cells were defective in the production of interleukin (IL)-2 and interferon gamma, but not IL-4. These data suggest that RIBP plays an important role in TCR-mediated signal transduction pathways and that its binding to Itk and Rlk/Txk may regulate T cell differentiation.

Involvement of the SHP-1 tyrosine phosphatase in regulation of T cell selection.

The selection events shaping T cell development in the thymus represent the outcome of TCR-driven intracellular signaling cascades evoked by Ag receptor interaction with cognate ligand. In view of data indicating TCR-evoked thymocyte proliferation to be negatively modulated by the SHP-1 tyrosine phosphatase, a potential role for SHP-1 in regulating selection processes was investigated by analysis of T cell development in H-Y TCR transgenic mice rendered SHP-1 deficient by introduction of the viable motheaten mutation or a dominant negative SHP-1-encoding transgene. Characterization of thymocyte and peripheral T cell populations in H-Y TCR-viable motheaten mice revealed TCR-evoked proliferation as well as the positive and negative selection of H-Y-specific thymocytes to be enhanced in these mice, thus implicating SHP-1 in the negative regulation of each of these processes. T cell selection processes were also augmented in H-Y TCR mice carrying a transgene driving lymphoid-restricted expression of a catalytically inert, dominant-negative form of SHP-1. SHP-1-negative effects on thymocyte TCR signaling were not influenced by co-cross-linking of the CD28 costimulatory and/or CTLA-4 inhibitory receptors and appear, accordingly, to be realized independently of these comodulators. These observations indicate that SHP-1 raises the signaling threshold required for both positive and negative selection and reveal the inhibitory effects of SHP-1 on TCR signaling to be cell autonomous. The demonstrated capacity for SHP-1 to inhibit TCR-evoked proliferation and selection indicate SHP-1 modulatory effects on the magnitude of TCR-generated signal to be a key factor in determining the cellular consequences of TCR-ligand interaction.

CD5 expression is developmentally regulated by T cell receptor (TCR) signals and TCR avidity.

Recent data indicate that the cell surface glycoprotein CD5 functions as a negative regulator of T cell receptor (TCR)-mediated signaling. In this study, we examined the regulation of CD5 surface expression during normal thymocyte ontogeny and in mice with developmental and/or signal transduction defects. The results demonstrate that low level expression of CD5 on CD4(-)CD8(-) (double negative, DN) thymocytes is independent of TCR gene rearrangement; however, induction of CD5 surface expression on DN thymocytes requires engagement of the pre-TCR and is dependent upon the activity of p56(lck). At the CD4(+)CD8(+) (double positive, DP) stage, intermediate CD5 levels are maintained by low affinity TCR-major histocompatibility complex (MHC) interactions, and CD5 surface expression is proportional to both the surface level and signaling capacity of the TCR. High-level expression of CD5 on DP and CD4(+) or CD8(+) (single positive, SP) thymocytes is induced by engagement of the alpha/beta-TCR by (positively or negatively) selecting ligands. Significantly, CD5 surface expression on mature SP thymocytes and T cells was found to directly parallel the avidity or signaling intensity of the positively selecting TCR-MHC-ligand interaction. Taken together, these observations suggest that the developmental regulation of CD5 in response to TCR signaling and TCR avidity represents a mechanism for fine tuning of the TCR signaling response.

Induction of antigen-specific hyporesponsiveness by transplantation of hemopoietic cells containing an MHC class I transgene regulated by a lymphocyte-specific promoter.

We explored a novel approach to tolerance induction by the transplantation of bone marrow (BM) cells (BMCs) that themselves do not express a foreign histocompatibility Ag, but which give rise to mature lymphocytes that do so. Lines of transgenic (FVB) mice were generated that contained an MHC class I Dd cDNA regulated by a CD2 promoter. Because the CD2 promoter is lymphocyte-specific and activated relatively late in lymphocyte ontogeny, Dd is expressed on most mature lymphocytes in the periphery but only on developing B cells in the BM of transgenic mice. Transgenic BMCs are tolerogenic and reproducibly engraft in nontransgenic mice using a conditioning regimen that is nonpermissive for the engraftment of conventional (MHC promoter) Dd-transgenic BMCs. Engrafted BMCs generate transgene-expressing lymphocytes and confer a state of Ag-specific hyporesponsiveness on the host that is primarily attributable to a peripheral mechanism. The strategies by which tolerance can be optimized in this system are discussed.

Comparison of EGS4 and MCNP Monte Carlo codes when calculating radiotherapy depth doses.

The Monte Carlo codes EGS4 and MCNP have been compared when calculating radiotherapy depth doses in water. The aims of the work were to study (i) the differences between calculated depth doses in water for a range of monoenergetic photon energies and (ii) the relative efficiency of the two codes for different electron transport energy cut-offs. The depth doses from the two codes agree with each other within the statistical uncertainties of the calculations (1-2%). The relative depth doses also agree with data tabulated in the British Journal of Radiology Supplement 25. A discrepancy in the dose build-up region may by attributed to the different electron transport algorithims used by EGS4 and MCNP. This discrepancy is considerably reduced when the improved electron transport routines are used in the latest (4B) version of MCNP. Timing calculations show that EGS4 is at least 50% faster than MCNP for the geometries used in the simulations.

Mice deficient in nuclear factor (NF)-kappa B/p52 present with defects in humoral responses, germinal center reactions, and splenic microarchitecture.

p52 is a subunit of nuclear factor (NF)-kappa B transcription factors, most closely related to p50. Previously, we have shown that p52, but not p50 homodimers can form transactivating complexes when associated with Bcl-3, an unusual member of the I kappa B family. To determine nonredundant physiologic roles of p52, we generated mice deficient in p52. Null mutant mice were impaired in their ability to generate antibodies to T-dependent antigens, consistent with an absence of B cell follicles and follicular dendritic cell networks in secondary lymphoid organs, and an inability to form germinal centers. Furthermore, the splenic marginal zone was disrupted. These phenotypes are largely overlapping with those observed in Bcl-3 knockout animals, but distinct from those of p50 knockouts, supporting the notion of a physiologically relevant complex of p52 homodimers and Bcl-3. Adoptive transfer experiments further suggest that such a complex may be critical in accessory cell functions during antigen-specific immune reactions. Possible roles of p52 and Bcl-3 are discussed that may underlie the oncogenic potential of these proteins, as evidenced by recurrent chromosomal translocations of their genes in lymphoid tumors.

Critical roles for the Bcl-3 oncoprotein in T cell-mediated immunity, splenic microarchitecture, and germinal center reactions.

Chromosomal translocations of bcl-3 are associated with chronic B cell lymphocytic leukemias. Previously, we have shown that Bcl-3, a distinct member of the I kappa B family, may function as a positive regulator of NF-kappa B activity, although its physiologic roles remained unknown. To uncover these roles, we generated Bcl-3-deficient mice. Mutant mice, but not their littermate controls, succumb to T. gondii owing to failure to mount a protective T helper 1 immune response. Bcl-3-deficient mice are also impaired in germinal center reactions and T-dependent antibody responses to influenza virus. The results reveal critical roles for Bcl-3 in antigen-specific priming of T and B cells. Altered microarchitecture of secondary lymphoid organs in mutant mice, including partial loss of B cells, may underlie the immunologic defects. The implied role of Bcl-3 in maintaining B cells in wild-type mice may related to its oncogenic potential.

Delayed maturation of CD4- CD8- Fc gamma RII/III+ T and natural killer cell precursors in Fc epsilon RI gamma transgenic mice.

Fc epsilon RI gamma (gamma) is a member of a group of related proteins (the zeta-family dimers) that function as signal-transducing components of both Fc receptors and the T cell antigen receptor (TCR). Analysis of gamma expression during fetal thymus ontogeny revealed that it is expressed in early thymocytes, before the initiation of clonotypic TCR-alpha and TCR-beta gene rearrangement but is down-regulated in most adult thymocytes. To explore a possible role for gamma in thymocyte development, we generated transgenic mice in which this protein was overexpressed at all stages of ontogeny. Overexpression of gamma inhibited the maturation of T cells as well as natural killer (NK) cells. The developmental effects were transgene dose related and correlated with markedly delayed maturation of fetal CD4-CD8- FcRII/III+ thymocytes, cells thought to include the progenitors of both T and NK cells. These results suggest that the zeta and gamma chains serve distinctive functions in thymocyte development and indicate that Fc receptor(s) may play an important role in regulating the differentiation of early progenitor cells within the thymus.

Cloning and characterization of PTP-K1, a novel nonreceptor protein tyrosine phosphatase highly expressed in bone marrow.

A novel nonreceptor protein tyrosine phosphatase (PTP), PTP-K1, was identified using a consensus polymerase chain reaction-based approach. The full length cDNA encompasses an open reading frame of 1362 base pairs, predicting a protein of 453 amino acid residues with a molecular mass of 54 kDa. The PTP domain is located in the N-terminal portion of the molecule and shares approximately 50% amino acid identify with two other nonreceptor PTPs: PEP and PTP-PEST. PTP-K1 is preferentially expressed in mouse bone marrow with transcripts of 1.7 kb, 1.9 kb and 3.5 kb. The 1.7 kb transcript was also detected in kidney, lung and ovary. The PTP domain of PTP-K1 was expressed as a fusion protein in bacteria and had intrinsic PTP catalytic activity. Indirect immunofluorescence microscopy in COS-7 cells showed that PTP-K1 was localized to the cytoplasm. Ptp-k1 was mapped to mouse chromosome 1, and was closely linked to the interleukin-1 receptor gene. The high level expression of PTP-K1 mRNA in bone marrow suggests that PTP-K1 may be involved in signal transduction in growth and differentiation of hematopoietic cells.

Murine txk: a protein tyrosine kinase gene regulated by T cell activation.

To identify genes involved in signal transduction pathways that regulate T cell activation and development, murine fetal thymocytes were screened for expression of protein tyrosine kinase family members by the polymerase chain reaction. Using this approach, a non-receptor protein tyrosine kinase, txk, was identified and cloned. Tsk is expressed in thymocytes as early as fetal day 13.5 and its expression at the mRNA level continues throughout development. Txk transcripts are present in thymocytes, peripheral T cells and mast cell lines, but are not detectable in B cell macrophage/monocyte cell lines or in non-hematopoietic fetal or adult tissues. In both thymocytes and T cells, txk transcripts are down-regulated after activation with PMA and ionomycin, concanavalin A or T cell receptor cross-linking. Sequence analysis indicates that txk contains SH2, SH3 and kinase catalytic domains and belongs to the tec family of cytoplasmic protein tyrosine kinases which includes tec, itk and btk. Its unique N-terminus contains a proline-rich region, but unlike the other tec family members, does not contain a pleckstrin homology domain. The restricted expression pattern of txk and its regulation by T cell activation make it an excellent candidate for involvement in signal transduction during thymocyte development.

Targeting of the T-cell receptor zeta-chain gene in embryonic stem cells: strategies for generating multiple mutations in a single gene.

The T-cell receptor zeta chain is a member of a family of related proteins that play a critical role in coupling cell-surface receptors to intracellular signaling pathways. To study the role of zeta chain in T-cell ontogeny, we generated targeted mutations of the zeta-chain gene in murine embryonic stem cells. The mutant alleles are predicted to result either in a null phenotype or in the synthesis of a truncated protein capable of supporting T-cell-receptor surface expression but deficient in transmembrane signaling. Both of these targeting events were recovered in a single electroporation experiment with either coelectroporation or a combination deletion/truncation construct. Our results suggest that similar approaches could be used to generate multiple single mutations, modifications of more than one site within a gene, or subtle alterations that rely upon coconversion with the selectable marker gene.