Ten-Eleven Translocation 1 and 2 Enzymes Affect Human Skin Fibroblasts in an Age-Related Manner.

Ten-eleven translocation (TET) enzymes catalyze the oxidation of 5-methylcytosine (5mC), first to 5-hydroxymethylcytosine (5hmC), then to 5-formylcytosine (5fC), and finally to 5-carboxycytosine (5caC). Evidence suggests that changes in TET expression may impact cell function and the phenotype of aging. Proliferation, apoptosis, markers of autophagy and double-strand DNA break repair, and the expression of Fibulin 5 were assessed by flow cytometry in TET1 and TET2-overexpressing fibroblasts isolated from sun-unexposed skin of young (23-35 years) and age-advanced (75-94 years) individuals. In cells derived from young individuals, TET1 overexpression resulted in the inhibition of proliferation and apoptosis by 37% (p = 0.03) and 24% (p = 0.05), respectively, while the overexpression of TET2 caused a decrease in proliferation by 46% (p = 0.01). Notably, in cells obtained from age-advanced individuals, TETs exhibited different effects. Specifically, TET1 inhibited proliferation and expression of autophagy marker Beclin 1 by 45% (p = 0.05) and 28% (p = 0.048), respectively, while increasing the level of γH2AX, a marker of double-strand DNA breaks necessary for initiating the repair process, by 19% (p = 0.04). TET2 inhibited proliferation by 64% (p = 0.053) and increased the level of γH2AX and Fibulin 5 by 46% (p = 0.007) and 29% (p = 0.04), respectively. These patterns of TET1 and TET2 effects suggest their involvement in regulating various fibroblast functions and that some of their biological actions depend on the donor's age.

An Anhydrous Sodium Chloride Skin Preservation Model for Studies on Keratinocytes Grafting into the Wounds.

BACKGROUND: Human skin is needed for covering large body areas lost by trauma. The shortcomings of contemporary methods of skin storage are limited preservation time and high immunogenicity if allogeneic. METHODS: We investigated whether long-lasting skin preservation in anhydrous sodium chloride (NaCl) may be the source of keratinocytes (KCs) for transplantation. Dehydrated skin fragments were preserved for a time frame from 1 week to 12 months. Then, skin fragments were rehydrated, and KCs were isolated. The viability of KCs was assessed in viability/cytotoxicity test. NaCl-preserved KCs were cultured for 7 days and transplanted to the dorsum of SCID mice. RESULTS: The morphology of NaCl-preserved KCs was unaltered. KCs from all epidermal layers could be identified. All grafts were accepted by the recipients. Transplanted KCs: synthesized keratins 10 and 16 expressed antigens specific for stem cells and transient-amplifying cells, and remained HLA-I-positive. Moreover, they expressed the proliferative marker PCNA. Cells isolated from transplants remained viable and produced enzymes. CONCLUSIONS: Transplantation of KCs obtained from human skin and stored in anhydrous NaCl may be considered for the closure of extensive skin wounds. The originality of this method consists of an effective storage procedure and easy preparation of keratinocytes for transplantation.

A unique case of diagnosis of a heterotopic pregnancy at 26 weeks - case report and literature review.

BACKGROUND: Heterotopic pregnancy (HP) is a rare condition when at least two pregnancies are present simultaneously at different implantation sites and only one located in the uterine cavity. The majority of cases are diagnosed in the first trimester. CASE PRESENTATION: We present a unique case of HP diagnosed at 26 weeks of spontaneous pregnancy in a patient without any relevant risk factors. We performed an extensive review of HP cases from MEDLINE (PUBMED) published in English between 2005-2019 to prove this case's uniqueness. A 24-year-old woman presented because of threatened preterm birth. Despite treatment, pain aggravated, without progression of labor. An emergency ultrasound exam revealed free fluid in the abdominal cavity. Suspicion of active bleeding prompted the medical team to perform an exploratory laparotomy. The surgery team found a ruptured heterotopic pregnancy. This was an unexpected cause of nontraumatic hemoperitoneum at such advanced gestational age. The postoperative period was uneventful, and the intrauterine pregnancy continued to term. The final review included 86 out of 124 records. A total number of 509 cases were identified, but not all of them had complete data. The maximum reported gestational age at the time of diagnosis was 16 weeks of pregnancy, while our case became symptomatic and was diagnosed at 26 weeks of pregnancy. CONCLUSIONS: Regardless of pregnancy age, HP can be a cause of hemoperitoneum, and it should be included in the differential diagnosis of acute abdomen in the second trimester.

Alterations in 5hmC level and genomic distribution in aging-related epigenetic drift in human adipose stem cells.

Aim: To clarify mechanisms affecting the level and distribution of 5-hydroxymethylcytosine (5hmC) during aging. Materials & methods: We examined levels and genomic distribution of 5hmC along with the expression of ten-eleven translocation methylcytosine dioxygenases (TETs) in adipose stem cells in young and age-advanced individuals. Results: 5hmC levels were higher in adipose stem cells of age-advanced than young individuals (p = 0.0003), but were not associated with age-related changes in expression of TETs. 5hmC levels correlated with population doubling time (r = 0.62; p = 0.01). We identified 58 differentially hydroxymethylated regions. Hypo-hydroxymethylated differentially hydroxymethylated regions were approximately twofold enriched in CCCTC-binding factor binding sites. Conclusion: Accumulation of 5hmC in aged cells can result from inefficient active demethylation due to altered TETs activity and reduced passive demethylation due to slower proliferation.

Cancer seeding contributes to intestinal anastomotic dehiscence.

BACKGROUND: Surgical wounds in cancer patients have a relatively high dehiscence rate. Although colon cancer resections are performed so as to include macroscopically non-involved tissues, some cancer cells can be present in the line of transection. The local healing process may facilitate proliferation of these localized cancer cells and the high cytokine concentration within the healing wound may also attract cancer cells from distant sites to migrate into the wound area. The growing tumor cells may then stretch the wound, hampering its contraction process. METHODS: The aim of the study was to monitor and compare, using immunohistochemical methods, the healing process of intestinal anastomosis in both normal rats and in rats with disseminated cancer (the CC531 colon cancer model). RESULTS: There was a significantly higher rate of anastomotic dehiscence in the group of rats with disseminated cancer, than in the group of normal rats. There were no significant differences between the two groups in the levels of mononuclear wound infiltration or of formation of connective tissue or new vessels. All anastomotic wounds in animals with disseminated cancer had abundant infiltrates of both migrating and proliferating cancer cells. CONCLUSIONS: We confirmed that the environment of a healing wound attracts cancer cells. Migration of cancer cells to the wound and centrifugal cancer proliferation may adversely affect the healing process and cause wound disruption.

Stewart-Treves syndrome angiosarcoma expresses phenotypes of both blood and lymphatic capillaries.

BACKGROUND: The development of angiosarcoma in oedematous tissue is referred to as Stewart-Treves syndrome (STS). This rare and fatal complication is associated with chronic post mastectomy lymphoedema and radiotherapy for breast cancer. Angiosarcoma spread is facilitated by the formation of blood vessels (angiogenesis) and lymph vessels (lymphangiogenesis). In the future antiangiogenic therapy may improve the poor outcome of current treatments. There was evidence that blocking the angiogenenesis would inhibit progression of angiosarcoma. It seems reasonable to hypothesize that blocking the lymphangiogenesis may yield similar results. Although angiosarcomas commonly derive from blood vessels, in case of STS angiosarcomas chronic lymphoedema may suggest its lymphatic origin. The goal of this study was to visualize interstitial space and lymphatics in the central and peripheral regions of STS angiosarcoma. METHODS: On tissue samples obtained from STS angiosarcoma we have performed: first colour stereoscopic lymphography to visualise the morphology of lymphatic vessels and extracellular spaces, second immunohistochemical staining specific for lymphatic vessels endothelium (LYVE-1) and blood endothelial cells (CD31, factor VIII) and prolymphangiogenic vascular endothelial growth factor (VEGF-C) for precise identification of lymphatic endothelia. STS angiosarcoma morphology was assessed by comparison of pictures obtained on lymphography, microscopy and confocal microscopy. RESULTS: STS angiosarcomas present heterogenous morphology with areas dominated by hemangiosarcoma and lymphangiosarcoma structures. STS angiosarcoma expressed phenotypes of both blood and lymphatic endothelia. LYVE-1 and VEGF-C is expressed by STS angiosarcoma and may be used to discriminate tumour differentiation. Morphology of lymphatic vessels and spaces in the tumour suggest absence of their normal lymphatic function. CONCLUSIONS: Our results confirmed both hemangio- and lymphangiogenic origin of STS angiosarcoma. Expression of VEGF-C makes STS angiosarcoma a good candidate for targeted antilymphangiogenic therapy. However, morphology of intratumoral lymphatics on colour lymphography suggested their impaired function, which can hamper drug distribution.

Lack of functioning intratumoral lymphatics in colon and pancreas cancer tissue.

There are controversial views as to whether intratumoral or peritumoral lymphatics play a dominant role in the metastatic process. Most clinical observations originate from studies of colon cancer. Colon contains mucosa and submucosa rich in lymphatics and with high lymph formation rate. This seems to be a prerequisite for easy metastasis of cancer cells to regional lymph nodes. However, there are other tissues as pancreas with a rudimentary lymphatic network where cancer metastasis formation is as intensive as in colon cancer. This contradicts the common notion that intratumor lymphatics play major role in metastases. We visualized interstitial space and lymphatics in the central and peripheral regions of colon and pancreas tumors using the color stereoscopic lymphography and simultaneously immunohistochemical performed stainings specific for lymphatic and blood endothelial cells. The density of open and compressed lymphatic and blood vessels was measured in the tumor core and edge. There were very few lymphatics in the colon and pancreas tumor core but numerous minor fluid "lakes" with no visible connection to the peritumoral lymphatics. Lining of "lakes" did not express molecular markers specific for lymphatic endothelial cells. Dense connective tissue surrounding tumor foci did not contain lymphatics. Peritumoral lymphatics were irregularly distributed in both types of tumor and only sporadically contained cells that might be tumor cells. Similar lymphoscintigraphic and histological pictures were seen in colon and pancreas cancer despite of different structure of both tissues. This suggests a uniform reaction of tissues to the growing cancer irrespective of the affected organ.

Spleen migrating dendritic cells primed with CC531 colon cancer antigen and LPS - is it a method to compromise liver metastases?

The anti-tumor vaccination is burdened by low recruitment rate of intravenously administered in vitro primed DC in liver metastases and lack of supplying them continuously in large numbers. Therefore, it seemed rational to create a model of in vivo vaccination with specifically primed splenic DC and cytotoxic T lymphocytes being continuously supplied to the liver vascular bed. The question we raised was whether anti-tumor immunized splenic DC flowing to liver metastases could adhere to and be cytotoxic to tumor cells. We immunized rats with CC531 tumor cells and stimulated them with Escherichia coli LPS. Subsequently, spleen DC-enriched population was isolated, its activation by LPS, adherence to CC531 cells and cytotoxicity were measured. Spleen cells home to the liver reaching it via splenic vein. These cells can be retrieved by simple washout of liver sinusoids (liver sinusoidal washout cells - LSWC). Their adherence to and cytotoxicity against CC531 cells were evaluated. Moreover, in vitro adherence of splenic DC-enriched cells and LSWC to CC531 liver tumor sections was measured. We found that in vivo immunization of splenic population containing DC, NK cells and lymphocytes with CC531 cells and stimulation with LPS activated these cells but did not significantly increase the cytotoxicity against CC531 cells. There was also no increase in cytotoxicity of LSWC. Adhesion of splenic DC and LWSC to liver CC531 metastases on cryosections was higher than to the adjacent liver tissue. However, it was more expressed on tumor stromal than neoplastic cells. The level of splenic Treg cells down-regulating immune response was found only slightly increased after immunization. Taken together, in the model of in vivo immunization against CC531 cells, low level of spleen DC and spleen-derived LSWC cytotoxicity as well as adherence rate to tumor cells were observed. More effective methods of immunizing splenic DC overcoming the suppressive mechanisms should be looked for.

[Microwave ablation of liver tumors as a new instrument for minimally invasive liver surgery]. / Mikrofalowa ablacja guzów watroby jako nowe narzedzie w malo inwazyjnej chirurgii watroby.

Incidence of primary and secondary liver tumors is increasing. Hepatic resection remains the treatment of choice for hepatic tumors. For various reasons the vast majority of patients with liver tumors are not suitable for resection. These patients are candidates for several image-guided focal thermal ablative therapies as alternatives to resection. Currently available ablative techniques include cryotherapy, radiofrequency ablation (RFA), microwave ablation (MWA), laser ablation, high-intensity focused ultrasound ablation (HIFU), and ethanol injection. Presently RFA is most widely heat-based technology used for treatment of liver malignancies due to its availability, efficacy and low complication rates. However, RFA can be time-consuming and associated with higher recurrence rates in larger lesions. MWA is a new thermal ablative technique that uses electromagnetic energy to produce coagulation necrosis. MWA has several advantages over RFA such as an improved convection profile, consistently higher intratumoral temperatures, larger ablation volumes, faster ablation times, and the option of using multiple antennae simultaneously. We report our first experience using MWA and a Coviden Valleyab 915 MHz generator for ablation of liver tumor with respect to our previous experience with RFA. Further this study reviews current literature on the RFA and MWA for the treatment of the liver malignances. In our opinion although experience is limited MWA appears to be a safe and effective technology for hepatic tumor ablation and in some cases may be superior alternative to RFA.

Surgical resection for persistent seroma, following modified radical mastectomy.

BACKGROUND: Seroma formation following modified radical mastectomy with axillary lymph node dissection for breast cancer is a most common wound complication. In our experience seroma occurs in approximately 50% of patients undergoing mastectomy. Postmastectomy seromas usually vanishes within a few weeks after operation. CASE PRESENTATION: In this report we present the case of a 73 year old woman who had undergone mastectomy with axillary lymph node dissection for breast cancer, complicated by lymphorrhea and formation fibrous encapsulated seroma resistant to conservative treatment which required surgical resection. CONCLUSION: We stand in opinion that in some cases of prolonged seromatous effusion with confirmed formation of thick walled reservoir the operation with resection and closure of supplying regional lymph vessels may be the best treatment, if possible preceded by arm lymphoscyntygraphy.

[The role of immune system in colon metastasis. Lymphangiogenesis or lymphedema in cancer tissue]. / Rola ukladu chlonnego w tworzeniu przerzutów raka jelita grubego. Limfangiogeneza czy limfedema w tkance nowotworowej.

The extent of lymph node metastasis is a major determinant for the staging and the prognosis of most human malignancies. Although the clinical significance of lymph node involvement is well documented, molecular mechanisms that promote tumor spread into the lymphatic or blood vascular systems and widespread dissemination are not well understood. Although there is a large body of evidence that newly visualized lymphatics facilitate formation of metastases, it remains unclear whether these are "new" or simply pre-existing dilated vessels. High level of permeability of tumor blood capillaries brings about high tissue fluid and lymph formation. The physical forces but not the putative cancer-produced VEGF C may be responsible for more lymphatics seen around cancer than in normal tissue. The main question to be answered is: are there morphologic and functional differences between newly formed and pre-existing intra- or peri-tumoral lymphatics? In our experience specimens of gastric and colon cancer revealed presence of peri-tumoral but not intra-tumoral lymphatics. Tumor tissue contained numerous tissue fluid "lakes" communicating with lymphatics. We speculate that increased production of lymph in the tumor tissue caused by high blood capillary permeability brings about dilatation of the interstitial space and peri-tumoral lymphatics. Excessive lymph flow may drag tumor cells. This article is reviews current literature on the role of angiogenesis and lymphangiogenesis in cancer metastasis with respect to own research.

[The role of sentinel node biopsy in breast cancer surgery]. / Rola biopsji wezla wartowniczego w leczeniu raka sutka.

Mastectomy with axillary lymph node dissection remains the routine surgical treatment of breast cancer in Poland. Lymph edema of the upper extremity is one of the major long-term complications of axillary dissection. Axillary lymph node status is the most valuable prognostic indicator and decision factor on adjuvant chemotherapy or radiotherapy for breast cancer patients. Level I and II axillary lymph node dissection provides prognostic information, maintains local control in the axilla and determines the need for adjuvant systemic treatment, but it is also associated with 30% rate of lymph edema. Multiple studies confirm that sentinel lymphadenectomy accurately stages cancer advancement and is associated with less morbidity than axillary dissection. Over 40% of breast cancer patients in Japan are submitted to breast conserving therapy with sentinel node biopsy. In our opinion sentinel node biopsy may be accepted as an alternative staging procedure for the axilla in breast cancer. Sentinel node biopsy is especially valuable tool for breast cancer patients undergoing breast sparing surgery (IIA), due to excellent cosmetic outcome, minimal morbidity and high degree of histological accuracy associated with the procedure. This article reviews current literature in breast conserving therapy and sentinel node biopsy. Author would like to thank to Professor Kenji Ogawa, Chairman of Surgical Department of Tokyo Women's Medical University Daini Hospital, Professor Fujio Kasumi Chief of Breast Surgical Department of Cancer Institute Hospital and the Japan Society for the Promotion of Science for the scientific support during research visit in Tokyo.