Multiplex In Situ Hybridization in the Study of Acute Kidney Injury : Multiplex In Situ Hybridization in AKI.

Recently developed in situ hybridization (ISH) methods, such as RNAscope™, have greatly expanded the accessibility and usefulness of ISH in biomedical research. Among many other advantages over traditional ISH, these newer methods enable the simultaneous use of multiple probes, including combination with antibody or lectin staining. We herein illustrate the application of RNAscope™ multiplex ISH in the study of the adapter protein Dok-4 in acute kidney injury (AKI). Specifically, we used multiplex ISH to define the expression of Dok-4 and some of its putative binding partners, together with nephron segment markers, as well as markers of proliferation and tubular injury. We also illustrate the use of QuPath image analysis software to perform quantitative analyses of multiplex ISH. Furthermore, we describe how these analyses can exploit the uncoupling of mRNA and protein expression in a knockout (KO) mouse created by CRISPR/CAS9-mediated frame shift to carry out highly focused molecular phenotyping studies at the single-cell level.

Concentrations of SP-A and HSP70 are associated with polarization of macrophages in pleural effusions of non-small cell lung cancer.

Damage-associated molecular pattern (DAMP) molecules can initiate an immune response through Toll-like receptors (TLRs). DAMPs are released from cells as a response to the extracellular danger and can be by-products of tissue damage. In cancer microenvironment necrotic cells release debris which has potency to become DAMPs. Non-small cell lung cancer (NSCLC) is often accompanied by pleural effusion (PE), which contains a variety of DAMPs. Surfactant protein A (SP-A) and heat shock protein 70 (Hsp70) are important DAMPs in the respiratory tract. The aim of this study was to determine a correlation between SP-A or Hsp70 and development of PE in the course of NSCLC. Moreover, we aimed to determine relationships between DAMPs and certain humoral factors associated with formation and persistence of PE as well as pleural-residing macrophages. In 34 PE samples, we estimated concentration of SP-A, Hsp70, IL-6, IL-18, G-CSF, M-CSF, SCF, SDF1α, VEGF as well as the fraction of macrophages and their pattern of polarization. We have found correlations between the concentration of the SP-A and Hsp70 and the percentage of PE-derived macrophages, also between concentrations of SP-A and Hsp70, and cytokines which participate in inflammation and processes involved in remodeling of extracellular matrix (ECM). Our data indicate an important role of SP-A during the development of PE associated with NSCLC. We suggest that measurement of concentration level of SP-A can be helpful in the course of diagnosis of malignant PE associated with NSCLC.

Local interleukin-2 and interleukin-12 therapy of bovine ocular squamous cell carcinomas.

Interleukin-2 and interleukin-12 have been used independently to successfully treat the induced and the spontaneous tumours in animals. This trial was done to determine if a combination of IL-2 and IL-12 in the treatment of spontaneous bovine ocular squamous cell carcinomas (BOSCC) would be more successful than IL-2 or IL-12 therapy by themselves. For this trial, we selected 25 BOSCC tumours seen on Holstein Fresian cows in Beatrice, Zimbabwe. The cows were randomly assigned to a treatment group of 5 days of IL-2 (200,000 U/day), 5 days of IL-12 (0.5 microg/day) or 5 days of IL-2 (200,000 U/day) and IL-12 (0.5 microg/day). At 20 months after treatment, the IL-2 therapy group had 63% complete regressions; the combination group had 38% complete regressions, which were significantly higher than the IL-12 group, which had 0% complete regressions at 20 months, despite having 29% complete regressions at 6 months. These results show that IL-2 therapy by itself and in combination with IL-12 is more successful than IL-12 by itself. However, combination therapy does not improve the outcome in comparison to IL-2 as a single therapy. It also proves that IL-2 is consistently successful in the therapy of BOSCC with over 60% complete regression, which corresponds to a number of other studies we have done on IL-2 therapy of BOSCC [Rutten, V.P.M.G., Klein, W.R., De Jong, W.A., Misdorp, W., Den Otter, W., Steerenberg, P.A., De Jong, W.H., Ruitenberg, E.J., 1989. Local interleukin-2 therapy in bovine ocular squamous cell carcinoma. A pilot study. Cancer Immunol. Immunother. 30, 165--169; Stewart, R.J.E., Hill, F.W.G., Masztalerz, A., Jacobs, J.J.L., Koten, J.W., Den Otter, W., 2003. Local low dose interleukin-2 therapy of bovine ocular squamous cell carcinomas in cattle in Zimbabwe, submitted for publication; Den Otter, W., Hill, F.W.G., Klein, W.R., Koten, J.W., Steerenberg, P.A., De Mulder, P.H.M., Rutten, V.P.M.G., Ruitenberg, E.J., 1993. Low doses of interleukin-2 can cure large bovine ocular squamous cell carcinoma. Anticancer Res. 13, 2453-2455; Den Otter, W., Hill, F.W.G., Klein, W.R., Koten, J.W., Steerenberg, P.A., De Mulder, P.H., Rhode, C., Stewart, R., Faber, J.A., Ruitenberg, E.J., 1995. Therapy of bovine ocular squamous cell carcinoma with local doses of interleukin-2: 67% complete regressions after 20 months of follow-up. Cancer Immunol. Immunother. 41, 10-14].

Treatment with IL-2 and IL-12 inhibits tumour cell division in SL2 lymphoma.

We examined which mechanism plays a dominant role in the rejection of solid SL2 lymphoma treated with locally applied IL-2 and/or IL-12. This treatment resulted in about 80% cures. There was a moderate influx of leukocytes in the tissue surrounding tumours; yet these cells failed to invade the solid tumours. Potentially cytotoxic cells were not observed in close proximity to areas of tumour cell death, indicating that cell-mediated cytotoxicity is not an important mechanism of tumour rejection in this model. Similarly, inhibition of blood vessel growth and/or blood vessel injury could be ruled out as mechanisms, since tumour rejection was not accompanied by decreased angiogenesis or blood vessel injury. We did observe that many tumour cells die via apoptosis or necrosis and that tumour cell division in cytokine-treated mice is inhibited. In conclusion, IL-2/IL-12-mediated tumour rejection in solid SL2 lymphoma is mainly due to a shifted balance between tumour cell death and tumour growth caused by inhibition of proliferation, rather than to direct cell cytotoxicity or destruction of blood vessels.

Synaptotagmin 3 deficiency in T cells impairs recycling of the chemokine receptor CXCR4 and thereby inhibits CXCL12 chemokine-induced migration.

Synaptotagmins regulate vesicle trafficking and fusion of vesicles with membranes - processes that have been implicated in cell migration. We therefore hypothesized that synaptotagmins play a role in T-cell migration. Amongst synaptotagmins 1-11, we found synaptotagmin 3 (SYT3) to be the only one that is expressed in T cells. CXCR4-triggered migration was inhibited by antisense synaptotagmin 3 mRNA and by the isolated C2B domain, known to impair oligomerization of all synaptotagmins, but not by a C2B mutant that binds Ca(2+) but does not block oligomerization. The C2B domain also blocked CXCR4-triggered actin polymerization and invasion. However, CXCR4-dependent adhesion in flow was not affected. Surprisingly, we found that little or no SYT3 is present near the plasma membrane but that it is mainly localized in multivesicular bodies, which also contained much of the CXCR4. Impaired SYT3 function blocked CXCR4 recycling and thus led to reduced surface levels of CXCR4. Migration was restored by overexpression of CXCR4. We conclude that STT3 is essential for CXCR4 recycling in T cells and thereby for the maintenance of high CXCR4 surface levels required for migration.

Presence of cytotoxic B220+CD3+CD4-CD8- cells correlates with the therapeutic efficacy of lymphoma treatment with IL-2 and/or IL-12.

Cancer treatment with IL-2 and IL-12 is thought to work via enhancement of proliferation and activity of T cells and NK cells. Incubation of cytotoxic T lymphocytes (CTLs) and NK cells with IL-2 and/or IL-12 results in propagation of a distinct cell type called lymphokine-activated killers (LAK) characterized by increased lytic activity against many tumor types. Here we address the question whether cytokine therapy may be efficient in treatment of a LAK-insensitive tumor and, if so, which cell type, other than classic LAK cells, is responsible for tumor cell killing. We used DBA/2 mice bearing metastasized SL2 lymphoma and treated them with locally applied IL-2 and /or IL-12 injections. We showed that IL-12 treatment is efficient, though there is a rather narrow range of effective doses because of toxicity. This toxicity may be alleviated by a single injection of IL-12 before treatment. Next, we showed that IL-12 synergistically enhances the efficacy of local IL-2 treatment. Moreover, our results indicate that the IL-2/IL-12-mediated therapeutic effect is greatest when it is given after establishment of an immune response to a tumor. Finally, we showed the existence of a unique population of lymphoid cells, namely B220+CD3+CD4-CD8-, at the site of tumor growth. These cells become highly cytotoxic to SL2 cells in mice treated with cytokines late (day 10-14) in the course of the immune response, but not in mice treated early (day 3-7), and cytotoxicity of this unique cell population correlates with the success of therapy.

Mechanisms of macrophage cytotoxicity in IL-2 and IL-12 mediated tumour regression.

IL-2 and IL-12 are promising anti-tumour agents. However, little attention has been paid to the role of macrophages during IL-2/IL-12 mediated tumour rejection. We studied the role of macrophages during IL-2/IL-12 mediated tumour rejection in DBA/2 mice bearing syngeneic SL2 lymphoma. Local treatment with IL-2 and IL-12 cured 85% of mice with severe metastasised tumour load. In vivo depletion studies showed that macrophages were required for the anti-tumour effect of IL-2 and IL-12. Macrophages could kill tumour cells both non-specifically and by antibody-dependent cellular cytotoxicity (ADCC). Treatment with IL-2, IL-12 or IL-2/IL-12 enhanced production of specific IgG1 immunoglobulins, while treatment with IL-12 and IL-2/IL-12 additionally induced IgG2a production. FcgammaRII and/or III were essential for ADCC expression after treatment with IL-2 and IL-12. These data show for the first time the essential role of macrophages during IL-2/IL-12 mediated tumour rejection and also suggest that IL-2 and IL-12 act via different mechanisms.