Patient-centred measurement of recovery from day-case surgery using wrist worn accelerometers: a pilot and feasibility study.

This pilot and feasibility study evaluated wrist-worn accelerometers to measure recovery from day-case surgery in comparison with daily quality of recovery-15 scores. The protocol was designed with extensive patient and public involvement and engagement, and delivered by a research network of anaesthesia trainees. Forty-eight patients recruited through pre-operative assessment clinics wore wrist accelerometers for 7 days before (pre-operative) and immediately after elective surgery (early postoperative), and again at 3 months (late postoperative). Validated activity and quality of recovery questionnaires were administered. Raw accelerometry data were archived and analysed using open source software. The mean (SD) number of valid days of accelerometer wear per participant in the pre-operative, early and late postoperative periods were 5.4 (1.7), 6.6 (1.1) and 6.6 (1.0) days, respectively. On the day after surgery, Euclidian norm minus one (a summary measure of raw accelerations), step count, light physical activity and moderate/vigorous physical activity decreased to 57%, 47%, 59% and 35% of baseline values, respectively. Activity increased progressively on a daily basis but had not returned to baseline values by 7 days. Patient questionnaires suggested subjective recovery by postoperative day 3 to 4; however, accelerometry data showed that activity levels had not returned to baseline at this point. All activity measures had returned to baseline by 3 months. Wrist-worn accelerometery is acceptable to patients and feasible as a surrogate measure for monitoring postoperative recovery from day-case surgery. Our results suggest that patients may overestimate their rate of recovery from day-case surgery, which has important implications for future research.

TNF-derived peptides inhibit tumour growth and metastasis through cytolytic effects on tumour lymphatics.

Tumour necrosis factor (TNF) is a multi-functional cytokine with profound and diverse effects on physiology and pathology. Identifying the molecular determinants underlying the functions and pathogenic effects of TNF is key to understanding its mechanisms of action and identifying new therapeutic opportunities based on this important molecule. Previously, we showed that some evolutionarily conserved peptides derived from TNF could induce cell death (e.g. apoptosis and/or necrosis), a feature of immune defence mechanisms shared by many vertebrates. In this study, we demonstrated that necrosis-inducing peptide P16 kills human glioblastoma cancer cells and primary human hepatoma or renal cancer cells isolated from patients who had not responded to standard treatments. Importantly, we show that the necrosis-inducing peptide P1516 significantly improves survival by inhibiting tumour metastasis in a 4T1 breast cancer syngeneic graft mouse model. Because the lymphatic system is an important metastatic route in many cancers, we also tested the effect of TNF-derived peptides on monolayers of primary human lymphatic endothelial cells (hDLEC) and found that they increased junctional permeability by inducing cytoskeletal reorganization, gap junction formation and cell death. Transmission electron microscopy imaging evidence, structural analysis and in-vitro liposome leakage experiments strongly suggest that this killing is due to the cytolytic nature of these peptides. P1516 provides another example of a pro-cytotoxic TNF peptide that probably functions as a cryptic necrotic factor released by TNF degradation. Its ability to inhibit tumour metastasis and improve survival may form the basis of a novel approach to cancer therapy.

Aselli Award Winner 2023: The Life and Times of a Lymphomaniac.

Winning a scientific prize is always a welcome honor and receiving the Aselli Award in 2023 was a particularly pleasant surprise. The following text provides a brief summary of the research carried out by my group in Oxford that led to the discovery of the lymphatic endo-thelial marker LYVE-1 and its emerging role as a pivotal receptor controlling the entry of immune cells and metastatic tumor cells to lymphatic capillaries in peripheral tissues. As a basic scientist and relatively late comer to the field of lymphology, my hope is that a closer partnership of basic and clinical research will help explain the intricate workings of the lymphatics and improve the picture for patients suffering from much neglected lymphatic disorders.

Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis.

Metastasis of breast cancer occurs primarily through the lymphatic system, and the extent of lymph node involvement is a key prognostic factor for the disease. Whereas the significance of angiogenesis for tumor progression has been well documented, the ability of tumor cells to induce the growth of lymphatic vessels (lymphangiogenesis) and the presence of intratumoral lymphatic vessels have been controversial. Using a novel marker for lymphatic endothelium, LYVE-1, we demonstrate here the occurrence of intratumoral lymphangiogenesis within human breast cancers after orthotopic transplantation onto nude mice. Vascular endothelial growth factor (VEGF)-C overexpression in breast cancer cells potently increased intratumoral lymphangiogenesis, resulting in significantly enhanced metastasis to regional lymph nodes and to lungs. The degree of tumor lymphangiogenesis was highly correlated with the extent of lymph node and lung metastases. These results establish the occurrence and biological significance of intratumoral lymphangiogenesis in breast cancer and identify VEGF-C as a molecular link between tumor lymphangiogenesis and metastasis.

VEGF-D promotes the metastatic spread of tumor cells via the lymphatics.

Metastasis to local lymph nodes via the lymphatic vessels is a common step in the spread of solid tumors. To investigate the molecular mechanisms underlying the spread of cancer by the lymphatics, we examined the ability of vascular endothelial growth factor (VEGF)-D, a ligand for the lymphatic growth factor receptor VEGFR-3/Flt-4, to induce formation of lymphatics in a mouse tumor model. Staining with markers specific for lymphatic endothelium demonstrated that VEGF-D induced the formation of lymphatics within tumors. Moreover, expression of VEGF-D in tumor cells led to spread of the tumor to lymph nodes, whereas expression of VEGF, an angiogenic growth factor which activates VEGFR-2 but not VEGFR-3, did not. VEGF-D also promoted tumor angiogenesis and growth. Lymphatic spread induced by VEGF-D could be blocked with an antibody specific for VEGF-D. This study demonstrates that lymphatics can be established in solid tumors and implicates VEGF family members in determining the route of metastatic spread.

Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3.

The lymphatic vasculature transports extravasated tissue fluid, macromolecules and cells back into the blood circulation. Recent reports have focused on the molecular mechanisms regulating the lymphatic vessels. Vascular endothelial growth factor (VEGF)-C and VEGF-D have been shown to stimulate lymphangiogenesis and their receptor, VEGFR-3, has been linked to human hereditary lymphedema. Here we show that a soluble form of VEGFR-3 is a potent inhibitor of VEGF-C/VEGF-D signaling, and when expressed in the skin of transgenic mice, it inhibits fetal lymphangiogenesis and induces a regression of already formed lymphatic vessels, though the blood vasculature remains normal. Transgenic mice develop a lymphedema-like phenotype characterized by swelling of feet, edema and dermal fibrosis. They survive the neonatal period in spite of a virtually complete lack of lymphatic vessels in several tissues, and later show regeneration of the lymphatic vasculature, indicating that induction of lymphatic regeneration may also be possible in humans.

Proteoglycan forms of the lymphocyte homing receptor CD44 are alternatively spliced variants containing the v3 exon.

The CD44 cell surface glycoprotein is expressed on a broad range of different tissues as multiple isoforms containing from one to ten alternatively spliced exons v1-v10 inserted within the extracellular domain. Differential glycosylation generates still further variability, yielding both N- and O-glycan-modified forms of CD44 in addition to proteoglycan-like variants containing chondroitin sulphate and heparan sulphate. These high molecular mass proteoglycan-like variants, previously identified in lymphocytes, melanomas, and keratinocytes have been implicated in cell-matrix adhesion, cell motility, and invasiveness. More recently, monocyte CD44 molecules presumed to carry glycosaminoglycan chains were shown to bind the chemokine MIP-1 beta (Tanaka, Y.,D. H. Adams, S. Hubscher, H. Hirano, U. Siebenlist, and S. Shaw. 1993. Nature (Lond). 361:79-82.) raising the intriguing possibility that proteoglycan-like CD44 variants might play a role in regulating inflammatory responses. Here we have investigated the molecular identity of these proteoglycan-like CD44 variants by generating a panel of recombinant CD44 isoforms using a novel cassette cloning strategy. We show that both chondroitin and heparan sulphate modifications are associated specifically with isoforms (CD44v3-10 and CD44v3,8-10) containing the v3 alternative exon which encodes a consensus motif SGXG for GAG addition. Other isoforms (CD44v10, CD44v8-10, CD44v7-10, and CD44v6-10) are shown to lack these GAG chains but to carry extensive O-glycan modifications, most likely within the mucin-like alternative exon inserts. We also demonstrate that the majority of endogenous GAG-modified CD44 isoforms present in epithelial cells constitute v3 isoforms thus establishing that in these cells the majority of proteoglycan-like CD44 variants are generated by alternative splicing. Finally we present evidence using transfected B lymphoma cells that the GAG-modified CD44 isoforms CD44v3-10 and CD44v3,8-10, unlike CD44H, bind only weakly to hyaluronan. Together with the demonstration in the accompanying paper (Bennett, K., D. G. Jackson, J.C. Simon, E. Tanczos, R. Peach, B. Modrell, I. Stamenkovic, G. Plowman, and A. Aruffo. 1995. J. Cell Biol. 128:687-698.), that CD44 molecules containing the v3 exon bind growth factors, these results highlight a new and potentially important role for CD44 alternative splicing in the control of cell-surface proteoglycan expression.

Regulation of CD44 binding to hyaluronan by glycosylation of variably spliced exons.

The hyaluronan (HA)-binding function (lectin function) of the leukocyte homing receptor, CD44, is tightly regulated. Herein we address possible mechanisms that regulate CD44 isoform-specific HA binding. Binding studies with melanoma transfectants expressing CD44H, CD44E, or with soluble immunoglobulin fusions of CD44H and CD44E (CD44H-Rg, CD44E-Rg) showed that although both CD44 isoforms can bind HA, CD44H binds HA more efficiently than CD44E. Using CD44-Rg fusion proteins we show that the variably spliced exons in CD44E, V8-V10, specifically reduce the lectin function of CD44, while replacement of V8-V10 by an ICAM-1 immunoglobulin domain restores binding to a level comparable to that of CD44H. Conversely, CD44 bound HA very weakly when exons V8-V10 were replaced with a CD34 mucin domain, which is heavily modified by O-linked glycans. Production of CD44E-Rg or incubation of CD44E-expressing transfectants in the presence of an O-linked glycosylation inhibitor restored HA binding to CD44H-Rg and to cell surface CD44H levels, respectively. We conclude that differential splicing provides a regulatory mechanism for CD44 lectin function and that this effect is due in part to O-linked carbohydrate moieties which are added to the Ser/Thr rich regions encoded by the variably spliced CD44 exons. Alternative splicing resulting in changes in protein glycosylation provide a novel mechanism for the regulation of lectin activity.

LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.

The extracellular matrix glycosaminoglycan hyaluronan (HA) is an abundant component of skin and mesenchymal tissues where it facilitates cell migration during wound healing, inflammation, and embryonic morphogenesis. Both during normal tissue homeostasis and particularly after tissue injury, HA is mobilized from these sites through lymphatic vessels to the lymph nodes where it is degraded before entering the circulation for rapid uptake by the liver. Currently, however, the identities of HA binding molecules which control this pathway are unknown. Here we describe the first such molecule, LYVE-1, which we have identified as a major receptor for HA on the lymph vessel wall. The deduced amino acid sequence of LYVE-1 predicts a 322-residue type I integral membrane polypeptide 41% similar to the CD44 HA receptor with a 212-residue extracellular domain containing a single Link module the prototypic HA binding domain of the Link protein superfamily. Like CD44, the LYVE-1 molecule binds both soluble and immobilized HA. However, unlike CD44, the LYVE-1 molecule colocalizes with HA on the luminal face of the lymph vessel wall and is completely absent from blood vessels. Hence, LYVE-1 is the first lymph-specific HA receptor to be characterized and is a uniquely powerful marker for lymph vessels themselves.

CD44 isoforms containing exon V3 are responsible for the presentation of heparin-binding growth factor.

Glycosaminoglycan-modified isoforms of CD44 have been implicated in growth factor presentation at sites of inflammation. In the present study we show that COS cell transfectants expressing CD44 isoforms containing the alternatively spliced exon V3 are modified with heparan sulfate (HS). Binding studies with three HS-binding growth factors, basic-fibroblast growth factor (b-FGF), heparin binding-epidermal growth factor (HB-EGF), and amphiregulin, showed that the HS-modified CD44 isoforms are able to bind to b-FGF and HB-EGF, but not AR. b-FGF and HB-EGF binding to HS-modified CD44 was eliminated by pretreating the protein with heparitinase or by blocking with free heparin. HS-modified CD44 immunoprecipitated from keratinocytes, which express a CD44 isoform containing V3, also bound to b-FGF. We examined whether HS-modified CD44 isoforms were expressed by activated endothelial cells where they might present HS-binding growth factors to leukocytes during an inflammatory response. PCR and antibody-binding studies showed that activated cultured endothelial cells only express the CD44H isoform which does not contain any of the variably spliced exons including V3. Immunohistological studies with antibodies directed to CD44 extracellular domains encoded by the variably spliced exons showed that vascular endothelial cells in inflamed skin tissue sections do not express CD44 spliced variants. Keratinocytes, monocytes, and dendritic cells in the same specimens were found to express variably spliced CD44. 35SO4(-2)-labeling experiments demonstrated that activated cultured endothelial cells do not express detectable levels of chondroitin sulfate or HS-modified CD44. Our results suggest that one of the functions of CD44 isoforms expressing V3 is to bind and present a subset of HS-binding proteins. Furthermore, it is probable that HS-modified CD44 is involved in the presentation of HS-binding proteins by keratinocytes in inflamed skin. However, our data suggests that CD44 is not likely to be the proteoglycan principally involved in presenting HS-binding growth factors to leukocytes on the vascular cell wall.

Adenoviral expression of vascular endothelial growth factor-C induces lymphangiogenesis in the skin.

The growth of blood and lymphatic vasculature is mediated in part by secreted polypeptides of the vascular endothelial growth factor (VEGF) family. The prototype VEGF binds VEGF receptor (VEGFR)-1 and VEGFR-2 and is angiogenic, whereas VEGF-C, which binds to VEGFR-2 and VEGFR-3, is either angiogenic or lymphangiogenic in different assays. We used an adenoviral gene transfer approach to compare the effects of these growth factors in adult mice. Recombinant adenoviruses encoding human VEGF-C or VEGF were injected subcutaneously into C57Bl6 mice or into the ears of nude mice. Immunohistochemical analysis showed that VEGF-C upregulated VEGFR-2 and VEGFR-3 expression and VEGF upregulated VEGFR-2 expression at 4 days after injection. After 2 weeks, histochemical and immunohistochemical analysis, including staining for the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), the vascular endothelial marker platelet-endothelial cell adhesion molecule-1 (PECAM-1), and the proliferating cell nuclear antigen (PCNA) revealed that VEGF-C induced mainly lymphangiogenesis in contrast to VEGF, which induced only angiogenesis. These results have significant implications in the planning of gene therapy using these growth factors.

Normal human tissues, in addition to some tumors, express multiple different CD44 isoforms.

At least 20 different isoforms of the human CD44 lymphocyte-homing receptor/hyaluronan receptor have been described to date that arise from the differential splicing of up to 10 alternative exons (termed v1-v10) encoding the membrane-proximal extracellular domain. Although numerous analyses at the mRNA level have indicated tissue-specific expression of CD44 variants, few analyses have been performed at the protein level because of limited availability of suitable monoclonal antibodies. Recently, however, exon-specific monoclonal antibodies have been generated using bacterial fusion proteins, and these have been reported to detect high levels of vCD44 containing the v6 exon on human tumors. Together with earlier evidence linking this particular exon with tumor metastasis in the rat, these latter experiments have led to the interpretation that v6 splice variants play a causative role in tumor dissemination. In this paper we describe the use of a new and comprehensive panel of CD44 exon-specific monoclonal antibodies generated against a recombinant CD44(v3-10)-immunoglobulin chimera to study vCD44 expression in a large number of normal and neoplastic tissues. We show that the expression of vCD44 varies greatly among different human tumors and that some express either very low levels of vCD44 or no CD44 at all. Furthermore, we demonstrate that expression is not limited to isoforms containing the v6 exon but includes variants carrying v3, v4/5, and v8/9. Additionally, normal epithelial tissues are shown to express considerable levels of these same vCD44 isoforms. Such results argue against a ubiquitous role for vCD44 isoforms in promoting tumor growth and metastasis.

LYVE-1 immunohistochemical assessment of lymphangiogenesis in endometrial and lung cancer.

AIMS/METHODS: Normal and malignant pulmonary and endometrial tissues were analysed for lymphatic vessels to assess the process of lymphangiogenesis and its role at these sites, using specific immunostaining for LYVE-1 and the panendothelial marker CD31. RESULTS: Lymphatics were clearly demonstrated in some normal tissues (myometrium, bronchial submucosa, and intestinal submucosa), but not in others (endometrium and alveolar tissue). LYVE-1 positive lymphatic vessels were detected at the tumour periphery of endometrial and lung carcinomas, but not within the main tumour mass. Double staining for LYVE-1 and the MIB1 proliferation marker revealed a higher proliferation index in lymphatic endothelial cells at the invading front of endometrial carcinomas, compared with myometrial areas distal to the tumour. Lung and endometrial carcinomas did not have an intratumorous lymphatic network. CONCLUSIONS: Although lymphangiogenesis may occur at the invading tumour front, incorporated lymphatics do not survive. Therefore, the dissemination of cancer cells through the lymphatics may occur by invasion of peripheral cancer cells into the adjacent normal lymphatics, or through shunts eventually produced at the invading tumour front as a consequence of active angiogenesis and lymphangiogenesis.

Angiogenesis and lymphangiogenesis in parathyroid proliferative lesions.

Angiogenesis and lymphangiogenesis are involved in tumoral growth and metastatic spread. There is little information on angiogenesis and no available data on lymphangiogenesis in parathyroid glands (PTG). Using immunohistochemistry for CD34, LYVE-1 (specific markers for vascular and lymphatic endothelium, respectively), vascular endothelial growth factor (VEGF)-A, VEGF-C, and fibroblast growth factor (FGF)-2, this study analyzes microvascular density (MVD), lymphatic vascular density (LVD), and expression of angiogenic and lymphangiogenic growth factors in 13 normal PTG, 77 parathyroid adenomas (PTA), and 17 primary parathyroid hyperplasia (PPH). MVD was higher in PPH and PTA, compared with PTG (P < 0.001). There was no difference in VEGF-A expression among groups. In contrast, FGF-2 expression was higher in PPH, compared with PTA and PTG (P < 0.0001). FGF-2 scores and MVD were significantly correlated (r = 0.43). LVD did not differ among groups, and VEGF-C expression was unrelated to LVD. There was no relationship between MVD and tumor behavior (adenoma size, PTH, or calcium). In conclusion, this study shows increased angiogenesis in parathyroid proliferative lesions compared with normal glands and suggests that FGF-2 is proangiogenic in parathyroid tissue. In PTA, tumor behavior is not related to angiogenic phenotype. This is the first demonstration of lymphatic vessels in PTG, but the lack of correlation with VEGF-C expression suggests that VEGF-C is not the primary lymphangiogenic factor.

Human lymphocyte antigen CD38 catalyzes the production of cyclic ADP-ribose.

The human lymphocyte antigen CD38 has been shown to share sequence homology with ADP-ribosyl cyclase, the enzyme that catalyzes the conversion of NAD+ to cyclic ADP-ribose (cADPR), a potent Ca(2+)-mobilizing agent. In this study COS1 cells from African Green Monkey kidney were transiently transfected with CD38 cDNA, inducing expression of authentic CD38 on the cell surface. We demonstrate that CD38 expressed in this manner can convert NAD+ to cADPR in the extracellular medium as assessed by Ca2+ release from sea-urchin egg microsomes.

TAP2 association with insulin-dependent diabetes mellitus is secondary to HLA-DQB1.

IDDM is known to be associated with genes of HLA complex, particularly alleles of HLA-DQ. The 40-kb TAP gene complex is located approximately 150 kb centrometric to the DQB1 locus. The TAP1-TAP2 protein heterodimer is required for normal expression levels of class I, molecules on the surface of cells. While present evidence implicates HLA-DQ as the major susceptibility locus in IDDM, as class I expression apparently plays a role in the progression of disease, the possibility exists that the association attributed to HLA-DQ is in fact due to an association with the TAP genes. Several studies have concluded that the alleles of TAP1 are not significantly associated with IDDM; this report concentrates on the more telomeric TAP2 locus. During this investigation, six previously described TAP2 alleles were identified in 208 normal Caucasians and 241 Caucasian diabetics. Sequence analysis of cDNA clones identified a seventh allele of TAP2, TAP2*F, which contains an arginine-to-cystine interchange at amino acid position 651. Overall, our results indicate only a modest association of IDDM with TAP2; however, the newly described TAP2*F allele was found to be significantly increased in a modest subset of our large diabetic population. These data, generated from the same population of controls and diabetics we previously studied at all other relevant MHC loci, provide additional evidence that the HLA susceptibility to IDDM maps to HLA-DQ.

Expression of a lymphatic endothelial cell marker in benign and malignant vascular tumors.

Tumors of endothelial cell origin are relatively common. Soft tissue tumors and numerous subtypes of benign and malignant vascular tumors have been described; the histogenesis of many of these tumors is uncertain, and distinguishing between benign and malignant vascular tumors, some of which express lymphatic endothelial cell markers, can be problematic. In the present study, immunophenotypic expression of a novel hyaluronan receptor (LYVE-1), which is expressed by endothelial cells of normal lymphatic vessels but not blood vessels, was determined in benign and malignant vascular tumors. It was found that, except in lymphangiomas, intramuscular hemangiomas, and Masson's hemangiomas, endothelial cells in benign blood vessel tumors (including capillary and cavernous hemangiomas, glomus tumors, pyogenic granulomas, and epithelioid hemangiomas) were negative for LYVE-1, and that all angiosarcomas and Kaposi's sarcomas were positive for LYVE-1. Expression of LYVE-1 and other lymphatic endothelial cell markers in relatively few vascular neoplasms has implications for the histogenesis of these lesions, and may prove useful in distinguishing angiosarcoma and Kaposi's sarcoma from most common benign vascular tumors.

New molecular markers for the study of tumour lymphangiogenesis.

The capacity of malignant tumours to metastasize to distant tissues presents a huge problem for the treatment of cancers using conventional surgical and cytotoxic drug therapies. One of the main routes for tumour spread is via the lymphatic vessels, an important conduit for tumours such as breast, lung and gastrointestinal tract that frequently colonize regional lymph nodes. In comparison with the vasculature however, little is known about the biology of tumour lymphatics, tumour lymphangiogenesis or the mechanisms that regulate entry and subsequent migration of tumour cells within lymphatic vessels. This situation has persisted because of the lack of specific molecular markers with which to visualize even normal lymphatics within tissues or to isolate lymphatic endothelial cells for in vitro experimental analysis. Just recently however, novel markers for lymphatic endothelial cells have been identified and their availability has revolutionized research in this field. In this article we highlight the main characteristics of these markers and review recent progress in their use to study tumour lymphangiogenesis.

Neuromyoblastoma in the rat.

We report three tumours arising in the brain stem or adjacent cranial nerves of the Alderly Park rat. Light microscopy with special stains, immunocytochemistry and electron microscopy supported the conclusion that both neuronal and myoid differentiation occurred in all neoplasms. This was shown by the presence in neural cells of axon-like cell processes and in myoblastic cells of striated myofibrils. Although medulloblastomas occur in man and rats the tumours reported here were not related to the cerebellum. We have termed this tumour neuromyoblastoma.

Fine morphology of the advancing front of cholesteatome--experimental and human.

The fine morphology of the advancing front of the cholesteatoma and the mucocutaneous juction of implanted skin in the feline bulla have been investigated. Inflammatory cell infiltration was frequently observed at the advancing front and the mucocutaneous junction. It is suggested that this frequent inflammation at this junction is due to the lack of a tight seal. In the advancing front, epidermal migrating cells appear to be lower spinous cells (or "suprabasalar" cells) rather than basal cells. It appears that the slender cell processes of the migrating cells make the initial attachment to the fibrin and/or basement membrane left behind by the degenerated epithelial cells as a result of inflammation and migrate along these structures, using them as a guide. Implanted skin in the cat's bulla failed to develop a pearl formation but frequently developed an epiboly, in which case the epidermis receded and was partly replaced by mucous membrane, and its stroma was heavily invaded by mucous epithelium, resembling tubular glands.