Cellulitis and bacteria in peripheral lymphedema.

Counterpoint to the article: Peculiar Clinical Features of Cellulitis in Peripheral Lymphedema, K. Suehiro, et al. Lymphology Vol 51, No 2, pp 47-53.

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.

Intrasplenic transplanted adult rat isolated hepatocyte fraction but not cholangiocytes forms bile canaliculi.

BACKGROUND: Hepatocyte transplantation (HT) has been performed in patients with liver-based metabolic disease and acute liver failure as a potential alternative to liver transplantation in countries in which ethical regulations do not allow organ transplantation. One of the problems remains that substances normally secreted by the surviving hepatocytes to bile cannot be removed because of lack of bile canaliculi. We found that ligation of the recipient's common bile duct in hepatocyte transplantation recipients is followed by formation of bile canaliculi. The question arose as to whether the signal released from the obstructed bile vasculature activated the transplanted hepatocytes (HC) or cholangiocyte (CH) to form bile canaliculi. METHODS: We transplanted separately isolated autologous HC and CH to spleens and observed the structural organization of the grafted cells. RESULTS: HC formed glycogen-rich clusters but not cords usually not attached to the CH of the new bile canaliculi. Separate clusters of bile canaliculi with keratin 7 and 19-positive and gamma-glutamyl transpeptidase-positive cells were observed. Transplanted CH remained keratin 7 and 19-positive and gamma-glutamyl transpeptidase positive but did not form canaliculi. CONCLUSIONS: The transplanted HC fraction may contain hepatic progenitor cells for cholangiocytes, but they become activated only under the condition of bile stasis by an as-yet undefined factor.

Lymphovenous microsurgical shunts in treatment of lymphedema of lower limbs: a 45-year experience of one surgeon/one center.

RATIONALE: The use of microsurgical lymphovenous shunts is one of the generally accepted treatments for limb lymphedema. AIM: The 45-year personal experience of one surgeon in indications, technique and results of lymphovenous shunt operations in lower limb lymphedema of varying etiology is presented. MATERIAL: One thousand three hundred patients were followed up in the period 1966-2011. Patients were classified into groups according to the etiology of lymphedema as postinflammatory/posttraumatic, postsurgical, idiopathic and hyperplastic. Decrease in limb circumference, heaviness and pain, and increase in joint flexing were evaluated. RESULTS: The most satisfactory results, reaching 80-100% improvement, were obtained in the congenital non-hereditary hyperplastic lymphedema group, with large lymphatics not previously damaged by infection. Results were also satisfactory in the group of cancer patients after iliac lymphadenectomy, reaching 80%. A less satisfactory outcome was observed in the postinflammatory group, not exceeding 30-40%. In idiopathic lymphedema results were satisfactory in only a few cases. CONCLUSIONS: Patients with lymphedema with local segmental obstruction but still partly patent distal lymphatics and without an active inflammatory process in the skin, subcutaneous tissue and lymph vessels present satisfactory results.

Magnetic resonance lymphography (MRL): point and counter-point.

Two preeminent lymphologists debate the findings, implications, interpretations, and value of magnetic resonance lymphography (MRL) in the evaluation of peripheral lymphedema. Their contrasting views are discussed in the context of different lymphatic imaging modalities including MRL, lymphoscintigraphy, and microscopic anatomy.

Chronic lower limb wounds evoke systemic response of the lymphatic (immune) system.

Wound healing should not be considered as a process limited only to the damaged tissues. It is always accompanied by an intensive local immune response and in advanced stages, the systemic lymphatic (immune) structure. In this review we present evidence from our own studies as well as pertinent literature on the role of skin and subcutaneous tissue lymphatics at the wound site and of transport of antigens along with collecting afferent lymphatics to the lymph nodes. We also speculate the role of lymph nodes in raising cohorts of bacterial and own tissue antigen-specific lymphocytes and their participation in healing and not infrequently evoking uncontrolled chronic immune reaction causing a delay of healing. It is also speculated as to why there is a rapid response of lymph node cells to microbial antigens and tolerance to damaged-tissue-derived antigens occurs.

Skin own bacteria may aggravate inflammatory and occlusive changes in atherosclerotic arteries of lower limbs.

AIM: Seroepidemiological studies have given rise to the hypothesis that microorganisms like Chlamydia pneumoniae (CP), Helicobacter pylori (HP), cytomegalovirus (CMV), HCV types 1 and 2, and bacteria involved in dental or other unspecified infection sites may initiate or maintain the atherosclerotic process in lower limb arteries. However, not much attention has been attached to the patient's own limb skin and deep tissues bacterial flora, activated in ischemic tissues. This flora may enhance the inflammatory and thrombotic process in the atherosclerotic arteries. Lower limb tissues are exposed to microorganisms from the environment (foot) and microbes on floating epidermal cells from the perineal and anal regions. The aim of this paper was to identify microbial cells and their DNA in perivascular tissues and arterial walls of lower limbs. METHODS: Bacterial cultures and PCR method for detection of 16sRNA and immunohistopathological staining for identification of immune cells infiltrating vascular bundles. RESULTS: 1) specimens of atherosclerotic calf and femoral arteries contained bacterial isolates and/or their DNA, whereas, in control normal cadaveric organ donors' limb arteries or patients' carotid arteries and aorta bacteria they were detected only sporadically; 2) lower limb lymphatics contained bacterial cells in 76% of specimens, whereas controls only in 10%; 3) isolates from limb arteries and lymphatics belonged in majority to the coagulase-negative staphylococci and S.aureus, however, other highly pathogenic strains were also detected; 4) immunohistopathological evaluation arterial walls showed dense focal infiltrates of granulocytes and macrophages. CONCLUSION: Own bacterial isolates can be responsible for dense neutrophil and macrophage inflitrates of atherosclerotic walls and periarterial tissue in lower limbs and aggravate the ischemic changes.

The pathophysiology of lymphedema - 2012.

Lymphedema of the limbs has become a frequent pathological condition after soft tissue inflammation, trauma, removal of lymph nodes in cancer and long-lasting ulcerations. Lymphatics draining the diseased tissues become occluded. Microsurgery helps in the formation of anastomoses and collaterals bypassing the obstruction site. Surgeons operating on the lymphatics should be aware of the tissue fluid/lymph formation mechanism, hydraulics of tissue fluid/lymph, tissue metabolism and waste material utilization, immune function in terms of elimination of microbial and tumor antigens and raising tolerance to own tissue antigens of injured tissues necessary for wound healing as well as classification of diseases of lymphatics. In this review we present the actual definition of the lymphatic system, how it is changed in lymphedema, and, in particular, tissue fluid/lymph biochemistry, pressure and flow, histopathology and tissue fluid location, and finally how to manage the most common complication dermato-lymphangioadenitis. Detailed knowledge of the anatomy of upper limb limphaties should prevent their damage and loss of function.

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.

The lymphovenous microsurgical shunts for treatment of lymphedema of lower limbs: indications in 2011.

The microsurgical lympho-venous shunts have become one of the generally accepted modalities in treatment of limb lymphedema. This review highlight the indications for this procedure after over 40 years. This study was based on the personal experience of one surgeon and on the review of the literature. Patients with postinflammatory, postsurgical, idiopathic and hyperplastic lymphedema of lower limbs were included in the study. Basing on the review of results of the last 40 years the contemporary indications are: 1) lymphedema with local segmental obstruction but still partly patent distal lymphatics seen on functional lymphoscintigraphy (standard walking or pneumatic compression) and without an active inflammatory process in the skin, subcutaneous tissue and lymph vessels (DLA-dermatolymphangioadenitis); 2) classified according the etiology of lymphedema, this operation can bring about satisfactory results in cases of hyperplastic, postsurgical and postinflammatory types of lymphedema, whereas primary idiopathic lymphedema of non-genetic type should be treated with conservative means, although in a small number of cases an improvement was observed after lympho-venous shunting as long as 10 years. Microsurgical lymph node or lymphatic vessel to vein shunts have their established position among the therapy modalities for lymphedema of lower limbs in a strictly defined group of patients using lymphoscintigraphic imaging.

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.

Where do lymph and tissue fluid accumulate in lymphedema of the lower limbs caused by obliteration of lymphatic collectors?

Obliteration of lymphatic collecting trunks of limbs by infective processes, trauma, oncologic surgery and irradiation bring about retention of lymph and tissue fluid in tissues. Knowledge as to where excess lymph is produced and accumulates as tissue fluid is indispensable for rational physical therapy. So far, this knowledge has been based on lymphoscintigraphic, ultrasonographic and MR images. None of these modalities provides distinct images of dilated lymphatics and fluid expanded tissue spaces in dermis, subcutis and muscles. Only anatomical dissection and histological processing of biopsy material can demonstrate the remnants of the lymphatic network and the sites of accumulation of mobile tissue fluid. We visualized and calculated the volume of the "tissue fluid and lymph" space in skin and subcutaneous tissue of foot, calf, and thigh in various stages of lymphedema, using special coloring techniques in specimens obtained during lymphatic microsurgical procedures or tissue debulking. When the collecting trunks were obliterated, lymph was present only in the subepidermal lymphatics, while mobile tissue fluid accumulated in the spontaneously formed spaces in the subcutaneous tissue, around small veins, and in the muscular fascia. Deformation of subcutaneous tissue by free fluid led to formation of interconnecting channels. In obstructive lymphedema caused by obliteration of collectors, lymph is present mainly in subepidermal lymphatics, and the bulk of stagnant tissue fluid accumulates in subcutis between fibrous septa and fat globules as well as above and underneath muscular fascia. These observations provide useful clues for designing pneumatic devices and rational manual lymphatic massage to move stagnant tissue fluid toward the non-swollen regions.

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.

Preservation of molecular structure of arterial grafts in pulverized sodium chloride: an experimental study.

There is a desperate need for preserved human arteries to be transplanted in infected areas of ischemic lower limbs. Cryopreserved arteries are fragile evoking strong allogeneic reactions. We have been searching for a preservation method that spares arterial wall structure and decreases its antigenicity. LEW and BN rat segments of aorta were harvested, immersed in pulverized dehydrated sodium chloride, and stored for 1 week to 3 months. Thereafter, they were desalinated and transplanted side-to-side to the abdominal aorta and side-to-side to the aorta-IVC (AV fistula). We performed staining for actin, elastic fibers, collagen (trichrome), CD31, endothelial cells (HIS52), MHC class II. Popliteal lymph node reactions were tested after subcutaneous implantation of an aortic fragment into the paw. We measured tensile strength and maximum intraluminal pressures. The preserved fragments of syngeneic aorta retained their cellular, stromal, and molecular structures. After transplantation, they did not evoke any reaction around the graft. The allogeneic fragments brought about only minor recipient responses, remaining patent and pulsatile for 3 months. The tensile strength and maximum intraluminal pressures did not significantly differ from freshly harvested, transplanted aortic segments.

Human splenic dendritic cells react differently to bacterial and allogeneic antigens.

We investigated the ability of bacterial (cells, LPS, and DNA) or allogeneic antigens to stimulate splenic dendritic cells (DCs) and expression of Toll-like receptors (TLRs), CD14 (co-functional molecule to TLR), CD83 (activation molecule on migrating DCs), CD123 (IL-3R specific for myeloid DC), Hsp60 and Hsp90 (heat shock proteins) involved in recognizing pathogen-associated molecular patterns (PAMP) and other pathogen recognition receptors (PRR) ligands. Allogeneic stimulation of DC TLRs was weak compared with that of bacterial products. This suggests that the highly conserved TLRs destined to react to bacterial products do not recognize donor products differing at the major histocompatibility complex (MHC) locus, at least using in vitro culture conditions.

Endogeneous sources of infection in transplant recipients.

The mammal organisms carry on their surfaces and in their tissues cohorts of microorganisms of various nature. There is a balance of interests and profits between the host and microbial inhabitants. The bacteria and fungi behave like comensals, colonizers, dormants, however, under certain, mostly unknown, conditions may evoke reaction of the host. This process is damaging both for the host and microbes. Large surgical trauma and allograft itself, as well as, immunosuppression create favorable conditions for imbalance between inhabiting microorganism and the recipient. The host flora and that transplanted with the organ graft become activated. Active combating of the proliferating bacteria with antibiotics becomes necessary. Our knowledge of the bacterial flora of the so called "sterile" tissues remains rudimentary. There is still a great deal of prejudice on the sterility of deep tissues e.g. muscles, fat tissue, etc. This review cumulates pertinent literature data on the microorganisms-host interactions. Our own findings on colonization of arteries and adjacent tissues are discussed in the context of atherosclerosis and grafting.

The effectiveness of long-acting penicillin (penidur) in preventing recurrences of dermatolymphangioadenitis(DLA) and controlling skin, deep tissues, and lymph bacterial flora in patients with "filarial" lymphedema.

Dermatolymphangioadenitis (DLA) is a common and serious complication of so-called "filarial" and bacterial non-filarial lymphedema of the limb, affecting skin, lymphatics and lymph nodes. In our previous studies, we demonstrated that more than 60% of patients revealed presence of bacterial isolates in deep tissues, tissue fluid and lymph from the lymphedematous limbs. The question remained open whether elimination or suppression of bacteria dwelling in lymphedematous tissues by administration of low doses of penicillin for long time periods would prevent recurrence of DLA attacks. In this study, we retrospectively evaluated a self/community-selected group of patients with lymphedema of the lower limbs with respect to the efficacy of long-acting penicillin in preventing episodes of DLA. There were no microfilariae or anti-filarial antibodies detected in the investigated group. The questions we asked were: (a) how effective is the benzathine penicillin in preventing recurrences of DLA attacks and (b) how does its long-term administration influence the bacterial spectrum of leg skin, deep tissues, lymph and lymph nodes and sensitivity to antibiotics. Two randomly selected groups of patients, receiving and not receiving penicillin during the same period of time, were compared. Evidently lower recurrence rate of DLA was observed in the treated group (p < 0.002). There was increased prevalence of cocci and gram-positive bacilli with a concomitant decrease of gram-negative bacilli on the foot and calf skin surface. Simultaneously, decreased prevalence of gram-positive cocci and gram-negative bacilli isolates in limb deep tissues and lymph was seen. No resistance to penicillin and other tested antibiotics developed in isolates from the skin surface, deep tissues and lymph. We conclude that long-lasting penicillin is effective in preventing recurrent DLA attacks.

Activation of splenic dendritic cells by bacterial and allogeneic antigens.

Allograft ischemia and cellular degradation accompanying rejection favor graft colonization by translocated microorganisms. Bacterial colonization adds to the graft destruction. The dendritic cells (DC) of allograft recipients engage in allogeneic and antibacterial reactions; they process and present to lymphocytes 2 types of antigens. This may lead to overstimulation of DCs that may nonspecifically intensify the rejection process. We investigated the effects of allogeneic and bacterial antigens on splenic DCs phenotypes. In vitro stimulation of a spleen DC-enriched population by E. coli, LPS, and CpG DNA brought about an increase in expression of OX6(+) (MHC class II) from 47.4% in the control cells to 65% in the E. coli-stimulated group (P < .05) and 85% in the LPS and CpGDNA groups (P < .05). Interestingly, a significant drop in the frequency of OX62(+) DC was observed after incubation with LPS. Allogeneic heart transplants brought about an increase of OX6(+) in DCs to 100% and a decrease of ED1(+) monocyte frequency. Simultaneously, an increase in expression of W3/13(+) T cells in DC-enriched splenic cells was observed. There was no significant change in the frequency of OX62(+) expression. Both types of antigens evoked splenic DC response; however, there were differences in the frequency of phenotype expression. Allogeneic but not bacterial antigens increased W3/13 antigen expression; the frequency of OX62(+) in cells decreased after LPS but not after bacterial stimulation.

Recipient immature dendritic cells do not prolong allograft survival.

Experimental studies on allogeneic transplantation have shown that recipient dendritic cells (DC) play a role in peripheral tolerance as well as in rejection of allografts. It is not known whether DCs exert their tolerogenic function in the graft or in recipient lymphoid tissue. To answer this question we created a chimeric heart model deprived of its own DCs and repopulated with recipient DCs. The rationale for this model was to observe whether recipient mature and immature DCs located in the graft attenuate recruitment and stimulation of recipient lymphocytes, subsequently prolonging graft survival. Vascularized bone marrow transplants from the prospective recipient to the lethally irradiated heart donor, which function for a period of 14 days, were used to replace donor DCs with prospective recipient either mature or immature DCs. Replacement of the donor heart with either of these cells did not prolong graft survival. The intragraft microchimerism did not mitigate the allogeneic rejection reaction.