Hyperkeratosis in human lower limb lymphedema: the effect of stagnant tissue fluid/lymph.

BACKGROUND: Hyperkeratosis of skin in lower limb lymphedema is one of the sequelae of tissue fluid/lymph (TF/L) stasis, but its mechanisms remain unknown. It is noteworthy, nonetheless, that human TF/L contains high levels of growth factors and cytokines, and may serve as the physiological environment for keratinocyte (KC) proliferation. OBJECTIVE: The aim of the study was to investigate the effect of human TF/L on human KC proliferation, differentiation and on the expression of epidermal stem cell markers on them. METHODS: KC were isolated from lymphedema and normal skin, and cultured for 1-14 days in TF/L with neutralized Interleukin 1ß, Interleukin 6, tumour necrosis factor α (TNF-α), keratinocyte growth factor (KGF) or tumour growth factor ß (TGF-ß). Alternatively, KC receptors for these factors were blocked. RESULTS: The number of KC cultured in TF/L was increased, as was the percentage of mitotic figures. There was a higher percentage of p63, CD29, Ki67, PCNA, CK6, CK17, CK16 and a lower of CK10, CK14, filaggrin and involucrin-positive KC. Neutralization of TF/L IL-1ß, IL-6, TNF-α and KGF as well as blockage of their receptors resulted in decreased percentage of mitotic KC. TGF-ß had a limited effect on KC proliferation. CONCLUSION: Hyperkeratosis in lymphedema may be the effect of a high concentration of cytokines in the stagnant TF/L tissue, but not because of presumed changes in the KC.

Transplantation of keratinocyte spore-like stem cells.

BACKGROUND: Wound granulation tissue should be covered by epidermal cells migrating from the basal layer of the epidermis or hair "bulge" of the wound edge. However, new epidermal islands are frequently formed on the granulation tissue remote from the wound edge. Thus, current theory of "bulge"-originating stem cells does not necessarily correspond to the histological pictures of the healing wound. We took imprints of a leg ulcer surface and found single dispersed, large nucleated cells, some of them in mitosis. These cells resembled those from epidermal spinosum layer. The question arouse as to whether these cells might be the "spore-like" stem cells creating epidermal island. We found similarly shaped cells among the keratinocyte preserved in pulverized sodium chloride as the only surviving population in culture and revealing enzymatic activity. The aim of this work was to study whether the population of human keratinocytes surviving sodium chloride preservation and transplanted to SCID mice may form epidermis. METHODS: The 12-month sodium chloride-preserved and cultured keratinocytes (KC) were transplanted to the wound on the dorsum of SCID mice for 14 and 21 days. RESULTS: Ninety-five percent of cultured KC were enzymatically active "large" cells; they did not express p63 and CD29 claimed as specific for stem cells, and they did not proliferate. Transplanted to the center of the wound, they formed small KC islands and became confluent after 14 days. CONCLUSIONS: The "large" epidermal keratinocytes survived the 12-month preservation in anhydrous sodium chloride. Transplanted to the wound, they formed epidermal islands of human phenotype. These cells may be the so-called "spore-like" stem cells.

Pathways of lymph and tissue fluid flow during intermittent pneumatic massage of lower limbs with obstructive lymphedema.

Questions remain on the use of sequential pneumatic compression including where does the fluid flow to and whether fluid can be moved to the non-swollen tissues of the hypogastrium and gluteal region? During pneumatic massage of the limb, we studied pathways of lymph and mobile tissue fluid flow using lymphoscintigraphy: a) from the calf and thigh across the inguinal region to the healthy non-swollen tissues of the hypogastrium and b) in the hypogastrium to the lateral and upper abdominal quadrants. To examine if there was effective fluid flow during pneumatic massage, plethysmographic flow measurements were also carried out. We demonstrated that: (i) pneumatic compression moved isotope in lymph remaining in functioning lymphatics and in tissue fluid in the interstitial space toward the inguinal region and femoral channel, (ii) there was no isotope crossing the inguinal crease or moving to the gluteal area, and (iii) isotope injected intradermally in the hypogastrium did not spread during manual massage to the upper and contralateral abdominal quadrants. In conclusion, intermittent pneumatic compression is effective in pushing mobile tissue fluid and relocating large fluid volumes toward the groin. However, the question that still remains is how to facilitate further flow toward the non-swollen tissues and thereby increase local absorption of fluid.

Lack of functioning lymphatics and accumulation of tissue fluid/lymph in interstitial "lakes" in colon cancer tissue.

There is controversy as to whether intratumoral or peritumoral lymphatics play a dominant role in the metastatic process. The knowledge of how and where exactly tumor cells enter lymphatics is important for therapeutic targeting either the tumor core or peritumoral tissue with drugs or radiation. The basic questions remain: what is the morphological structure of intra- and peritumoral interstitium and lymphatics; what is their hydraulic conductivity?; and do these local physical conditions allow detached tumor cells to migrate to lymphatics? Identification of lymphatics has been based on immunohistochemical staining of lymphatic endothelial cells. This method does not, however, show the tissue fluid filled interstitial space and the shape of minute lymphatic vessels in tumors. We visualized the interstitial space and lymphatics in the central and peripheral regions of tumors using our original method of color stereoscopic lymphography in translucent tissue fragments and simultaneously with immunohistochemical staining of lymphatic and blood endothelial cells. The density of open and compressed lymphatic and blood vessels was measured in the intratumoral "hot spots" and at tumor edge. Moreover, the intratumoral tissue hydraulic conductivity was measured to define force necessary for propelling tissue fluid to peritumoral lymphatics. We found very few rudimentary minor blind lymphatics in the tumor core and numerous minor fluid "lakes" in the interstitium with no visible connection to the peritumoral lymphatics. Lining of "lakes" did not express molecular markers specific for lymphatic endothelial cells. Ninety-five percent of structures of what looked like lymphatics had compressed lumen and the hydraulic conductivity was 3 powers of magnitude lower than in the adjacent non-tumoral tissue. It can be concluded that lack of functioning lymphatics in tumor foci manifested by accumulation of tissue fluid in "lakes," low fluid conductivity and compression of lymphatics by tumor cells, and proliferating connective tissue may hamper escape of tumor cells. The most favorable site of entry of tumor cells to lymphatics seems to be the interface of the tumor and surrounding tissue with open lymphatics.

The influence of tissue fluid/lymph cytokines and growth factors on human keratinocyte proliferation and differentiation.

Cultured keratinocytes (KC) are needed for transplantation to the surface of large burn wounds and ulcers. They can be cultured in artificial media. However, the yield is always limited, viability is low, and proliferation and migration after grafting are slow. The question arose whether tissue fluid/lymph, which is a natural humoral environment for epidermal and dermal cells, contains cytokine(s) specifically regulating KC proliferation and could be used to culture large numbers of cells for transplantation. Culturing of skin keratinocytes in dermal tissue fluid/lymph containing keratinocyte growth factor, interleukin-1beta, interleukin-6, tumor necrosis factor-alpha, and transforming growth factor-beta revealed its strong stimulatory effect on the expression of p63 stem cell marker and proliferation but not differentiation of KC. Neutralizing these cytokines with antibodies resulted in decreased percentages of mitotic figures. None of the individual cytokines showed a dominant effect on proliferation. This observation suggests that either there may be other (so far undetected) specific cytokines or that the proliferation and differentiation of keratinocytes is an effect of the combined action of all investigated cytokines.