Rajen Ramsamooj

Delivery of antioxidative enzyme genes protects against ischemia/reperfusion–induced liver injury in mice

Hepatic ischemia/reperfusion (I/R) injury is characterized by the generation of reactive oxygen species (ROS), such as superoxide anions and hydrogen peroxide. The aim of this study is to investigate whether antioxidative gene delivery by our polylipid nanoparticles (PLNP) is an effective approach for prevention of the injury. Polyplexes of extracellular superoxide dismutase (EC‐SOD) and/or catalase genes were injected via the portal vein 1 day prior to a warm I/R procedure in mice. The effects of the gene delivery were determined 6 hours after starting reperfusion. PLNP‐mediated antioxidative gene delivery led to a marked increase in human EC‐SOD and catalase gene expression in the liver. Liver superoxide dismutase (SOD) and catalase activity both increased approximately 10‐fold. Increased liver superoxide anion levels caused by the I/R procedure were reduced to normal levels by EC‐SOD gene delivery. The overexpression of these 2 antioxidative genes significantly suppressed the I/R‐induced elevation of serum alanine aminotransferase (ALT) levels, decreased liver malondialdehyde content, restored glutathione reserve, and improved liver histology. In conclusion, EC‐SOD or catalase gene delivery by PLNP resulted in high levels of the transgene activity in the liver, and markedly attenuated hepatic I/R injury. The protection is directly associated with elevated antioxidative enzyme activity as the result of the gene delivery. This novel approach may become a potential therapy to improve graft function and survival after liver transplantation. Liver Transpl 12:1869–1879, 2006.

Development of stable mixed T cell chimerism and transplantation tolerance without immune modulation in recipients of vascularized bone marrow allografts

A consistent majority (62.5%) of immunologically unmodified rat recipients transplanted with vascularized hind-limb bone marrow allografts across a semiallogeneic transplant barrier developed tolerance with absence of graft-versus-host disease. A minority of recipients (37.5%) demonstrated lethal GVHD. Transplantation tolerance in the majority was associated with the induction of stable low-level mixed T cell chimerism, including donor CD5+, CD4+, and CD8+ lymphocytes. Chimeras were specifically immune nonresponsive to host alloantigenic determinants. These results emphasized a potentially important mechanism for low-level stable mixed lymphoid chimerism (SMLC) in tolerance induction, independent of immune suppressive effects due to irradiation or immunopharmacologic intervention. These vascularized bone marrow transplantation (VBMT) results may establish the experimental foundation for a novel approach to stem cell transfer and bone marrow transplantation.

Near-infrared autofluorescence imaging for detection of cancer

Near-infrared autofluorescence imaging of tissues under long-wavelength laser excitation in the green and red spectral region complemented by cross-polarized elastic light scattering was explored for cancer detection. Various types of normal and malignant human tissue samples were utilized in this investigation. A set of images for each tissue sample was recorded that consisted of two autofluorescence images obtained under 532- and 632.8-nm excitation and light-scattering images obtained under linearly polarized illumination at 700, 850, and 1000 nm. These images were compared with the histopathology of the tissue sample. The experimental results indicated that for various tissue types, the intensity of the autofluorescence integrated over the 700 to 1000-nm spectral region was considerably different in cancer tissues than in that of the contiguous non-neoplastic tissues. This difference provided the basis for the detection of cancer and delineation of the tumor margins. Variations on the relative intensity were observed among different tissue types and excitation wavelengths.

Composite tissue allografts in rats: IV. Graft-versus-host disease in recipients of vascularized bone marrow transplants

This laboratory has used a composite tissue allograft model as a vehicle for studies on a new type of bone marrow transplant, the vascularized bone marrow transplant. The model consists of a rat hind limb transplant that incorporates integumentary musculoskeletal, and lymphopoietic tissues. These transplants, in comparison with conventional marrow transplants, have the advantage of providing a syngeneic microenvironment and immediate engraftment of both mature and progenitor hemopoietic cells at the time of transplantation. The characteristics of graft-versus-host disease were studied in this model. Lewis X Brown Norway F1 (LBN RT-1(1+n)) rats received hind limbs from Lewis (LEW RT-1(1)) donors (n = 19). Animals were observed daily for signs of graft-versus-host disease. Necropsies were performed. A minority of animals developed lethal disease (7 of 19 recipients) and demonstrated cachexia with concomitant histopathologic changes of the disease. Acute and chronic groups emerged with distinct clinical courses, which are similar to other models of this disease. Recipients of vascularized bone marrow transplants (limb transplants) showed clinical and histopathologic changes of the disease. The transplants may be used as a model of graft-versus-host disease in humans. Most interestingly, the transplant has a lower incidence of disease compared with other methods of bone marrow transplantation and represents an alternative to conventional bone marrow transplantation, which deserves further exploration. It may be possible to develop a new technique for bone marrow transplantation based on this surgical approach. It is proposed that the transfer of vascularized blocks of bone/marrow into prospective recipients as opposed to cellular bone marrow transplants may be preferable.

Spectroscopic detection of bladder cancer using near-infrared imaging techniques

High-contrast imaging of bladder cancer is demonstrated using near-infrared autofluorescence under long-wavelength laser excitation in combination with cross-polarized elastic light scattering. Fresh unprocessed surgical specimens obtained following cystectomy or transurethral resection were utilized and a set of images for each tissue sample was recorded. These images were compared with the histopathology of the tissue sample. The experimental results indicate that the intensity of the near-infrared emission as well as that of the cross-polarized backscattered light was considerably different in cancer tissues than in that of the contiguous nonneoplastic tissues, allowing an accurate delineation of a tumors margins.

Hepatoma SK Hep-1 Cells Exhibit Characteristics of Oncogenic Mesenchymal Stem Cells with Highly Metastatic Capacity

Background SK Hep-1 cells (SK cells) derived from a patient with liver adenocarcinoma have been considered a human hepatoma cell line with mesenchymal origin characteristics, however, SK cells do not express liver genes and exhibit liver function, thus, we hypothesized whether mesenchymal cells might contribute to human liver primary cancers. Here, we characterized SK cells and its tumourigenicity. Methods and Principal Findings We found that classical mesenchymal stem cell (MSC) markers were presented on SK cells, but endothelial marker CD31, hematopoietic markers CD34 and CD45 were negative. SK cells are capable of differentiate into adipocytes and osteoblasts as adipose-derived MSC (Ad-MSC) and bone marrow-derived MSC (BM-MSC) do. Importantly, a single SK cell exhibited a substantial tumourigenicity and metastatic capacity in immunodefficient mice. Metastasis not only occurred in circulating organs such as lung, liver, and kidneys, but also in muscle, outer abdomen, and skin. SK cells presented greater in vitro invasive capacity than those of Ad-MSC and BM-MSC. The xenograft cells from subcutaneous and metastatic tumors exhibited a similar tumourigenicity and metastatic capacity, and showed the same relatively homogenous population with MSC characteristics when compared to parental SK cells. SK cells could unlimitedly expand in vitro without losing MSC characteristics, its tumuorigenicity and metastatic capacity, indicating that SK cells are oncogenic MSC with enhanced self-renewal capacity. We believe that this is the first report that human MSC appear to be transformed into cancer stem cells (CSC), and that their derivatives also function as CSCs. Conclusion Our findings demonstrate that SK cells represent a transformation mechanism of normal MSC into an enhanced self-renewal CSC with metastasis capacity, SK cells and their xenografts represent a same relative homogeneity of CSC with substantial metastatic capacity. Thus, it represents a novel mechanism of tumor initiation, development and metastasis by CSCs of non-epithelial and endothelia origin.

Tissue imaging for cancer detection using NIR autofluorescence

Near IR imaging using elastic light scattering and tissue fluorescence under long-wavelength laser excitation are explored for cancer detection. Various types of normal and malignant human tissue samples were utilized in this investigation. A set of images of each tissue sample is recorded. These images are then compared with the histopathology of the tissue sample to reveal the optical fingerprint characteristics suitable for cancer detection. The experimental results indicate that the above approaches can help image and differentiate cancer form normal tissue.

Hyperspectral imaging of cells: toward real-time pathological assessment

The goal of this work is to develop micro-scale noninvasive photonic instrumentation and techniques that will enable real-time imaging and monitoring of microstructures and cells in tissues. We utilize a hyperspectral microscope to explore the differences of native fluorescence and polarized light scattering from cellular components using various excitation wavelengths. The key optical “signature” characteristics that differentiate the various cellular components are used to obtain composite images that highlight their presence and the relative concentration of various tissue chromophores. The sensitivity has been optimized in our specially designed instrumentation so that image acquisition times are very short for real-time application in a clinical setting. This technology is not invasive to the cells and therefore it can be used to monitor their function while they are still alive.

Mechanisms of alloimmune tolerance associated with mixed chimerism induced by vascularized bone marrow transplants

Rat limb allograft recipients represent surgically induced, immediately vascularized bone marrow transplant (VBMT) chimeras. The majority of these chimeras undergo tolerance while a minority develop graft versus host disease (GVHD). T-cell chimerism and associated mechanisms of cellular immune nonresponsiveness were investigated in tolerant VBMT chimeras. A strong correlation (p < 0.01) was observed between the clinical onset of GVHD and levels of donor T-cell chimerism approximating or greater than 50%. However, stable mixed chimerism was associated with tolerance. In conclusion, three major sequential mechanisms of immune nonresponsiveness were elucidated in tolerant VBMT chimeras over time and included development of nonspecific suppressor cells (which potentially represent natural suppressor cells), maturation of antigen-specific suppressor cell circuits, and eventually putative clonal inactivation.

Acute renal transplant injury and interaction between antithymocyte globulin and pooled human immunoglobulin.

Abstract:  The increasing number of highly sensitized patients awaiting renal transplantation has prompted the use of induction immunosuppression regimens including pooled human intravenous immunoglobulin (IVIG) combined with polyclonal anti‐lymphocyte sera. We report a case of a patient who received a live donor transplant after abrogation of donor‐specific positive cytotoxic crossmatch by IVIG. She developed early acute tubular injury associated with IVIG, mannitol, and hypertonic saline infusion. Furthermore, a possible interaction between IVIG and rabbit antithymocyte globulin (ATG) occurred, suggested by an increased number of peripheral CD3+ lymphocytes after initial rapid lymphodepletion. We suggest that IVIG‐associated nephrotoxicity should be considered in the differential diagnosis of early allograft dysfunction, and furthermore, that IVIG may interact with polyclonal antilymphocyte serum to affect the amount of lymphodepletion achieved.