Detection of adrenomedullin and nitric oxide in different forms of periodontal disease.

BACKGROUND AND OBJECTIVE: The multifunctional molecules adrenomedullin (AM) and nitric oxide (NO) are both involved in the host response to microbial challenge during periodontal disease. Whether they coexist in periodontal inflammation and if equally produced in the different forms of periodontal disease has not previously been investigated. The aims of this study were to describe the locations of AM and NO in healthy and inflamed gingival tissues and to determine and compare their levels in the gingival crevicular fluid and saliva of patients with gingivitis, chronic periodontitis and aggressive periodontitis. MATERIAL AND METHODS: AM and inducible nitric oxide synthase (iNOS) were immunolocalized in clinically healthy and inflamed gingival tissue sections. The cells expressing AM and iNOS were characterized using immunocytochemistry with different markers for macrophages [cluster differentiation (CD)68 and CD14)], dendritic cells (CD83), neutrophils [neutrophil gelatinase-associated lipocalin (nGAL)] and natural killer cells (CD56). In an initial study, the levels of AM and NO were also measured in samples of gingival crevicular fluid and saliva obtained from patients with a diagnosis of gingivitis (n = 9), chronic periodontitis (n = 9) and aggressive periodontitis (n = 9) using an ELISA and the nitrate/nitrite (NO metabolites) Griess assay, respectively. RESULTS: Low levels of AM- and iNOS-expressing cells were detected in healthy gingival tissues in comparison with three-fold higher levels of these cells in inflamed tissues. These cells were localized mainly in the epithelial layer but were also present in deeper connective tissue. AM and iNOS were co-localized in particular cells within inflamed tissues, namely CD68(+) (52%) and CD14(+) (36%) macrophages, but also in nGAL(+) neutrophils (16%) and CD83(+) dendritic cells (14%). Interestingly, AM and NO levels in saliva were both found to be higher (p < 0.01) in patients with aggressive periodontitis than in patients with chronic periodontitis or gingivitis. In contrast, in gingival crevicular fluid, the levels of NO showed marked differences among patients with chronic periodontitis, aggressive periodontitis and gingivitis (p < 0.01), and the levels of AM were higher (p < 0.01) in both chronic and aggressive periodontitis compared with gingivitis alone. CONCLUSION: The data presented demonstrate a functional linkage between AM and NO in periodontal disease, with salivary and gingival crevicular fluid levels possibly associated with different forms and severities of periodontal disease. Exacerbated production of both AM and NO in saliva suggests their potential use as salivary markers of aggressive periodontitis.

Regulation of adrenomedullin and nitric oxide production by periodontal bacteria.

OBJECTIVES: In periodontitis the host response to bacterial challenge includes activity of the multifunctional molecules adrenomedullin (AM) and nitric oxide (NO). The aim of this study was to investigate the role of periodontal bacteria in regulating the production of these molecules from cultured cells. MATERIAL AND METHODS: Regulation of AM and NO production from oral keratinocytes when challenged with culture supernatants from Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Veillonella atypica, Streptococcus salivarius and Candida albicans was examined. AM and NO were measured in cell culture supernatants using an enzyme-linked immunosorbent assay and the nitrate/nitrite (NO metabolites) Griess assay respectively. Cellular production of AM and inducible NO synthase was also analysed in target cells by immunofluorescence and Western blot analysis. The inter-relationship of AM and NO production were further investigated with macrophages. RESULTS: A. actinomycetemcomitans and C. rectus induced maximal levels of both AM and NO after 6 and 48 h respectively from oral keratinocytes. AM production in macrophages was upregulated in response to the NO donor S-nitrosoglutathione and partially blocked by the inducible NO synthase inhibitor, N(ω) -Nitro-l-arginine methyl ester hydrochloride. Likewise, NO production was increased upon exposure to AM, while the AM receptor antagonist AM 22-52 reduced the release of NO. CONCLUSIONS: Pathogens associated with aggressive periodontitis, A. actinomycetemcomitans and C. rectus, were more effective than those associated with chronic periodontitis, P. gingivalis and Prev. intermedia, and commensals, S. salivarius and V. atypica, as regards the upregulation of AM and NO production from oral keratinocytes. Interaction between these molecules was also demonstrated with macrophages. Understanding the coordinated regulation of AM and NO production in response to periodontal bacteria may identify ways to promote their protective effects and minimize destructive potential.

Effects of hydroxyapatite and PDGF concentrations on osteoblast growth in a nanohydroxyapatite-polylactic acid composite for guided tissue regeneration.

The technique of guided tissue regeneration (GTR) has evolved over recent years in an attempt to achieve periodontal tissue regeneration by the use of a barrier membrane. However, there are significant limitations in the currently available membranes and overall outcomes may be limited. A degradable composite material was investigated as a potential GTR membrane material. Polylactic acid (PLA) and nanohydroxyapatite (nHA) composite was analysed, its bioactive potential and suitability as a carrier system for growth factors were assessed. The effect of nHA concentrations and the addition of platelet derived growth factor (PDGF) on osteoblast proliferation and differentiation was investigated. The bioactivity was dependent on the nHA concentration in the films, with more apatite deposited on films containing higher nHA content. Osteoblasts proliferated well on samples containing low nHA content and differentiated on films with higher nHA content. The composite films were able to deliver PDGF and cell proliferation increased on samples that were pre-absorbed with the growth factor. nHA-PLA composite films are able to deliver active PDGF. In addition the bioactivity and cell differentiation was higher on films containing more nHA. The use of a nHA-PLA composite material containing a high concentration of nHA may be a useful material for GTR membrane as it will not only act as a barrier, but may also be able to enhance bone regeneration by delivery of biologically active molecules.

Effects of low-dose doxycycline on cytokine secretion in human monocytes stimulated with Aggregatibacter actinomycetemcomitans.

Doxycycline is an antibiotic used in the treatment of a variety of inflammatory conditions, including periodontitis. Apart from its antimicrobial properties, this drug also has independent anti-inflammatory effects at sub-antimicrobial doses. The present study aimed to investigate the effects of low-doses of doxycycline (LDD) on cytokine production by human monocytic cells challenged with the periodontal pathogen Aggregatibacter actinomycetemcomitans, for up to 6 h. The simultaneous regulation of 12 cytokines were measured by a Human Cytokine Array Kit. To validate the array findings, selected cytokines were also measured by enzyme-linked immunosorbant assay (ELISA). A. actinomycetemcomitans stimulated the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1ß, IL-6 and IL-8 by the cells after 6 h of challenge, and doxycycline significantly inhibited this effect. The kinetics of this regulation demonstrated an early (within 2 h) and significant (P<0.05) inhibition of pro-inflammatory cytokines, with a mild (0.5-fold) up-regulation of the anti-inflammatory cytokine IL-10. The results indicate that LDD acts as an anti-inflammatory agent in human monocytic cells stimulated with A. actinomycetemcomitans. This model provides clear evidence that some of the clinically proven benefits of LDD may be related to its ability to regulate inflammatory mediator release by monocytic cells. This property may contribute to the clinically proven benefits of this antibiotic as an adjunctive treatment for periodontitis.

The T1799A point mutation is present in posterior uveal melanoma.

An activating mutation in exon 15 of the BRAF gene is present in a high proportion of cutaneous pigmented lesions. Until recently this mutation had however only been identified in one case of posterior uveal melanoma. Despite this apparent lack of the BRAF mutation, inappropriate downstream activation of the Ras/Raf/MAPK pathway has been described in posterior uveal melanoma. Based on the already recognised morphological and cytogenetic heterogeneity in uveal melanoma, we hypothesised that the BRAF mutation may be present in uveal melanoma but only in some of the tumour cells. In this study, we analysed 20 ciliary body and 30 choroidal melanomas using a nested PCR-based technique resulting in the amplification of a nested product only if the mutation was present. This sensitive technique can identify mutated DNA in the presence of wild-type DNA. The mutation was identified in 4 of 20 (20%) ciliary body and 11 of 30 (40%) choroidal melanomas. Further analysis of separate areas within the same choroidal melanoma demonstrated that the mutation was not present in the entire tumour. In conclusion, the T1799A BRAF mutation is present in a proportion of posterior uveal melanomas but within these tumours the distribution of the mutation is heterogeneous.

Malignant transformation of murine fibroblasts by a human c-Ha-ras-1 oncogene does not require a functional epidermal growth factor receptor.

Although mutations in ras genes are thought to be important for the development of about 20% of human tumors, almost nothing is known about the way in which these mutations lead to cellular transformation. The known biochemical properties of the 21-kilodalton ras proteins suggest that they may behave as G proteins, regulating the proliferation of cells in response to growth factor stimulation of a receptor. Although the putative receptor(s) has not been identified, several lines of evidence, in particular the fact that rodent cell lines containing ras oncogenes produce transforming growth factor alpha, have suggested that the epidermal growth factor (EGF) receptor is involved in ras transformation. Here we show that murine fibroblasts with no EGF receptors can be transformed to a completely malignant phenotype with a mutated ras gene. It appears, therefore, that the EGF receptor is not required for ras-mediated transformation of these cells.

Hosts, seasonality and geographic distribution of the South African tortoise tick, Amblyomma marmoreum.

The tortoise tick Amblyomma marmoreum was collected from large numbers of reptiles and other animals during the course of numerous surveys conducted in South Africa. A total of 1229 ticks, of which 550 were adults, were recovered from 309 reptiles belonging to 13 species, with leopard tortoises, Geochelone pardalis being the most heavily infested. The 269 birds sampled harboured 4901 larvae, 217 nymphs and no adult ticks, and the prevalence of infestation was greatest on helmeted guineafowls, Numida meleagris. Only two larvae were recovered from 610 rodents, including 31 spring hares, Pedetes capensis, whereas 1144 other small mammals yielded 1835 immature ticks, of which 1655 were collected from 623 scrub hares, Lepus saxatilis. The 213 carnivores examined harboured 2459 ticks of which none were adult. A single adult tick and 6684 larvae and 62 nymphs were recovered from 656 large herbivores, and a total of 4081 immature ticks and three adults were collected from 1543 domestic animals and 194 humans. Adult male and female A. marmoreum were most numerous on reptiles during January and February, and larvae during March. The largest numbers of larvae were present on domestic cattle and helmeted guineafowls in the Eastern Cape Province during March or April respectively, whereas larvae were most numerous on helmeted guineafowls, scrub hares and the vegetation in north-eastern Mpumalanga Province during May. In both provinces nymphs were most numerous between October and December. Amblyomma marmoreum appears to be most prevalent in the western regions of the Western and Eastern Cape and Free State provinces, and the north-eastern regions of the Northern Cape, KwaZulu-Natal, Mpumulanga and Limpopo provinces.

Parasites of domestic and wild animals in South Africa. XLVII. Ticks of tortoises and other reptiles.

A total of 586 reptiles, belonging to 35 species and five subspecies, were examined in surveys aimed at determining the species spectrum and geographic distribution of ticks that infest them. Of these reptiles 509 were tortoises, 28 monitor or other lizards, and 49 snakes. Nine ixodid tick species, of which seven belonged to the genus Amblyomma, and one argasid tick, Ornithodoros compactus were recovered. Seven of the ten tick species are parasites of reptiles. Amongst these seven species Amblyomma marmoreum was most prevalent and numerous on leopard tortoises, Geochelone pardalis; Amblyomma nuttalliwas present only on Bell's hinged tortoises, Kinixys belliana; and most Amblyomma sylvaticum were collected from angulate tortoises, Chersina angulata. Amblyomma exornatum (formerly Aponomma exornatum) was only recovered from monitor lizards, Varanus spp.; most Amblyomma latum (formerly Aponomma latum) were from snakes; and a single nymph of Amblyomma transversale (formerly Aponomma transversale) was collected from a southern African python, Python natalensis. All 30 Namaqualand speckled padloper tortoises, Homopus signatus signatus, examined were infested with O. compactus. The seasonal occurrence of A. sylvaticum and the geographic distribution of this tick and of A. marmoreum, A. nuttalli, A. exornatum, A. latum and O. compactus are illustrated.

Mutant but not normal p21 ras elevates inositol phospholipid breakdown in two different cell systems.

Expression of oncogenic mutant p21 ras either in stably transformed NIH3T3 fibroblasts or transiently in COS-1 cells elevates the basal rate of inositol phosphate production. Additional mutations in the effector region or the carboxy terminal region which abolish transforming capacity on NIH3T3 cells block the effect of oncogenic mutant p21 ras on basal rates. Overexpression of normal (Gly12) p21 ras has no such effect on this second messenger signalling system. These differences between overexpressed normal p21 ras and oncogenic mutant p21 ras strongly suggest that the increased basal rate of inositol phosphate production is a direct consequence of constitutively activated p21 ras activity.

The murine homeobox gene Msx-3 shows highly restricted expression in the developing neural tube.

The mouse homeobox-genes Msx-1 and Msx-2 are expressed in several areas of the developing embryo, including the neural tube, neural crest, facial processes and limb buds. Here we report the characterisation of a third mouse Msx gene, which we designate Msx-3. The embryonic expression of Msx-3 was found to differ from that of Msx-1 and -2 in that it was confined to the dorsal neural tube. In embryos with 5-8 somites a segmental pattern of expression was observed in the hindbrain, with rhombomeres 3 and 5 lacking Msx-3 while other rhombomeres expressed Msx-3. This pattern was transient, however, such that in embryos with 18 or more somites expression was continuous throughout the dorsal hindbrain and anterior dorsal spinal cord. Differentiation of dorsal cell types in the neural tube can be induced by addition of members of the Tgf-beta family. Additionally, Msx-1 and -2 have been shown to be activated by addition of the Tgf-beta family member Bmp-4. To determine if Bmp-4 could activate Msx-3, we incubated embryonic hindbrain explants with exogenous Bmp-4. The dorsal expression of Msx-3 was seen to expand into more ventral regions of the neurectoderm in Bmp-4-treated cultures, implying that Bmp-4 may be able to mimic an in vivo signal that induces Msx-3.

Is carotid duplex scanning sufficient as the sole investigation prior to carotid endarterectomy?

Carotid endarterectomy (CEA) is the accepted treatment for certain patients who have had, or who are at risk of having, a stroke if they have a significant narrowing of the internal carotid artery. Rapid and accurate classification of the degree of stenosis is important as the benefit of surgery is highly dependent on this. The aim of this study was to assess whether the addition of angiography to duplex scanning resulted in a change in patient management in a unit where duplex scanning was used as the sole imaging investigation prior to CEA. The study population consisted of 64 patients with significant internal carotid artery stenosis on duplex scanning who were suitable for, and wished to be considered for, CEA. All patients underwent an angiogram. In this study 9 (14%) patients did not proceed to surgery on the basis of angiography and in a further 11 (17%) patients insufficient views of the distal vessel were obtained on duplex scanning. Three of these patients had extensive disease which excluded surgery. One patient experienced a transient ischaemic attack (TIA) at the time of angiography. In conclusion, this audit has highlighted the limitations in performing duplex scanning alone, and the costs that this can incur on the patient who may undergo an unnecessary operation. We cannot recommend duplex scanning as the sole investigation prior to CEA. There is need to evaluate the role of additional non-invasive carotid imaging such as magnetic resonance angiography or CT angiography in the assessment of these patients.

Culturing skin in vitro for wound therapy.

Current tissue-culture techniques enable keratinocytes from a small piece of skin to be grown into sheets of epithelium, or cultured keratinocyte grafts, that are suitable for treating wounds. Serial subculture enables rapid expansion of a cell population, such that grafts of a total area equivalent to that of the surface of an adult can be obtained from an initial skin biopsy of approximately 2 cm2 in under one month. In this article, the methods currently used for culturing keratinocytes, the search for a fully functional replacement for the dermal elements of skin, and the prospects for clinical development of these technologies in the near future are discussed.

Transcriptional regulators of steroidogenesis, DAX-1 and SF-1, are expressed in human skin.

DAX-1 and SF-1 are members of the orphan nuclear receptor superfamily that are critical regulatory components of the hypothalamic-pituitary-adrenal-gonadal axis. In adrenal and gonadal tissues they regulate the expression of the cytochrome P450 steroid hydroxylase genes, key mediators of steroidogenesis. The identification of a number of steroid hydroxylases in human skin prompted us to investigate the presence of DAX-1 and SF-1. Immuno histochemical analysis of human skin revealed a distinctive staining pattern for DAX-1 and SF-1 in skin and its appendages. Prominent staining for DAX-1 was confined to the epidermis, sebaceous glands, sweat glands, and outer root sheath of the hair follicle with weaker expression in the inner root sheath, matrix cells, and dermal papilla cells. Similarly, SF-1 was also detected in the epidermis but displayed a scattered nuclear pattern across all layers. SF-1 immunoreactivity was also detected in the exocrine glands and was stronger than DAX-1 in the inner root sheath, matrix cells, and dermal papilla cells. Co-localization of DAX-1 and SF-1 was demonstrated by immunocytochemistry in the HaCaT keratinocyte cell line, primary keratinocytes, preadipocytes, and dermal papilla cells. Reverse transcriptase-polymerase chain reaction analysis demonstrated the expression of DAX-1 and SF-1 mRNA in whole human skin and Western analysis also confirmed the presence of DAX-1 protein in skin-derived cells. Our investigations demonstrate that two important regulators of steroidogeneisis are present in human skin and its appendages. These transcription factors may have a role in cutaneous steroidogenesis and thus be involved in hair follicle cycling or pathologies associated with steroids. Further studies are needed to determine the functional roles of DAX-1 and SF-1 in human skin.

Contrasting localization of c-Myc with other Myc superfamily transcription factors in the human hair follicle and during the hair growth cycle.

The mammalian hair follicle is a highly dynamic skin appendage that undergoes repeated cycles of growth and regression, involving closely co-ordinated regulation of cell proliferation, differentiation, and apoptosis. The Myc superfamily of transcription factors have been strongly implicated in the regulation of these processes in many tissues. Using immunohistochemistry, we have investigated the patterns of c-Myc, N-Myc, Max, and Mad1-4 expression at different stages of the human hair growth cycle. N-Myc, Max, Mad1, and Mad3 immunoreactivity was detected in the epidermis and the epithelium of both anagen and telogen hair follicles. Three distinct patterns of hair follicle c-Myc immunoreactivity were observed. In the infundibulum, c-Myc staining was predominantly in the basal layers, with little detectable immunoreactivity in the terminally differentiating suprabasal layers; this pattern was similar to that seen in the epidermis. In contrast, c-Myc expression in the follicle bulb was found both in the proliferating germinative epithelial cells and in the terminally differentiating matrix cells that give rise to the hair fiber. Finally, intense c-Myc immunoreactivity was detected in the bulge region of the outer root sheath. Using the C8/144B antibody as a bulge marker, we confirmed that c-Myc immunoreactivity in the outer root sheath correlates with the putative hair follicle stem cell compartment. c-Myc expression in the bulge was independent of the hair growth cycle stage. Our data suggest that Myc superfamily members serve different functions in separate epithelial compartments of the hair follicle and may play an important role in determining cell fate within the putative stem cell compartment.

Patterns of proliferation and apoptosis during murine hair follicle morphogenesis.

In this study, we have correlated cutaneous apoptosis and proliferation in neonatal mice during hair follicle morphogenesis. We have applied a novel triple- staining technique that uses Ki67 immunoreactivity as a marker of proliferation as well as TUNEL and Hoechst 33342 staining as apoptosis markers. We have also assessed the immunoreactivity of interleukin-1 beta-converting enzyme, caspase 1, a key enzyme in the execution of apoptosis, and of P-cadherin, which has been suggested as a key adhesion receptor in segregating proliferating keratinocytes. The TUNEL data were systematically compared with high resolution light microscopy and transmission electron microscopy data. Virtually all keratinocytes of the developing hair bud were strongly Ki67(+), suggesting that the hair bud is not an epidermal invagination but primarily the product of localized keratinocyte proliferation. As hair follicle development advanced, three distinct foci of proliferation became apparent: the distal outer root sheath around the hair canal, the mid outer root sheath, and the proximal hair matrix. Of these proliferating hair follicle keratinocytes only defined subsets expressed P-cadherin. TUNEL(+) cells in the hair follicle were not found before stage 5 of murine hair follicle morphogenesis. During the early stages of hair follicle development, interleukin-1 beta-converting enzyme immunoreactivity was present on all keratinocytes, but virtually disappeared from the proximal hair follicle epithelium later on. High resolution light microscopy/transmission electron microscopy revealed scattered and clustered apoptotic keratinocytes in all epithelial hair follicle compartments throughout hair follicle development, including its earliest stages. This highlights striking differences in the demarcation of apoptotic hair follicle keratinocytes between the TUNEL technique and high resolution light microscopy/transmission electron microscopy and suggests a role for apoptosis in sculpting the hair follicle even during early hair follicle development.

A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages.

Numerous strains of mice with defined mutations display pronounced abnormalities of hair follicle cycling, even in the absence of overt alterations of the skin and hair phenotype; however, in order to recognize even subtle, hair cycle-related abnormalities, it is critically important to be able to determine accurately and classify the major stages of the normal murine hair cycle. In this comprehensive guide, we present pragmatic basic and auxiliary criteria for recognizing key stages of hair follicle growth (anagen), regression (catagen) and quiescence (telogen) in C57BL/6NCrlBR mice, which are largely based on previous work from other authors. For each stage, a schematic drawing and representative micrographs are provided in order to illustrate these criteria. The basic criteria can be employed for all mouse strains and require only routine histochemical techniques. The auxiliary criteria depend on the immunohistochemical analysis of three markers (interleukin-1 receptor type I, transforming growth factor-beta receptor type II, and neural cell-adhesion molecule), which allow a refined analysis of anatomical hair follicle compartments during all hair cycle stages. In contrast to prior staging systems, we suggest dividing anagen III into three distinct substages, based on morphologic differences, onset and progression of melanogenesis, and the position of the dermal papilla in the subcutis. The computer-generated schematic representations of each stage are presented with the aim of standardizing reports on follicular gene and protein expression patterns. This guide should become a useful tool when screening new mouse mutants or mice treated with pharmaceuticals for discrete morphologic abnormalities of hair follicle cycling in a highly reproducible, easily applicable, and quantifiable manner.

Human beta defensin-1 and -2 expression in human pilosebaceous units: upregulation in acne vulgaris lesions.

A rich residential microflora is harboured by the distal outer root sheath of the hair follicle and the hair canal - normally without causing skin diseases. Although the basic mechanisms involved in the development of inflammation during acne vulgaris remain unclear, microbial agents might play an important role in this process. In this study we have analyzed by in situ hybridization and immunohistochemistry the expression patterns of two antimicrobial peptides, human beta defensin-1 and human beta defensin-2, in healthy human hair follicles as well as in perilesional and intralesional skin of acne vulgaris lesions such as comedones, papules, and pustules. Strong defensin-1 and defensin-2 immunoreactivity was found in all suprabasal layers of the epidermis, the distal outer root sheath of the hair follicle, and the pilosebaceous duct. Marked defensin-1 and defensin-2 immunoreactivity was also found in the sebaceous gland and in the basal layer of the central outer root sheath including the bulge region. The majority of acne biopsies displayed a marked upregulation of defensin-2 immunoreactivity in the lesional and perilesional epithelium - in particular in pustules - and a less marked upregulation of defensin-1 immunoreactivity. The upregulation of beta-defensin expression in acne vulgaris lesions compared to controls suggests that beta-defensins may be involved in the pathogenesis of acne vulgaris.

Antiviral pathway activation in patients with chronic fatigue syndrome and acute infection.

Gene expression of key enzymes in 2 antiviral pathways (ribonuclease latent [RNase L] and RNA-regulated protein kinase [PKR]) was compared in 22 patients with chronic fatigue syndrome (CFS), 10 patients with acute gastroenteritis, and 21 healthy volunteers. Pathway activation in the group of patients with infections differed significantly from that of the other 2 groups, in whom there was no evidence of upregulation. Therefore, assay of activation is unlikely to provide the basis for a diagnostic test for CFS.

ERF-2, the human homologue of the murine Tis11d early response gene.

A human cDNA specifying a member of the Tis11 early response gene family was cloned and sequenced. The human gene differs from its mouse homologue by encoding an additional 97 amino acids at its C-terminal end. The sequence has transactivation-like motifs, an unusual Cys-Ser-Ala-rich motif and displays sequence similarity at the extreme C-terminal end with another Tis11 family member, ERF-1.

The use of collagen or fibrin gels for the assay of human neutrophil chemotaxis.

Neutrophil leucocytes are known to migrate actively into 3-dimensional gels of collagen or fibrin. In this paper, we have used such gels to study chemotaxis of human blood neutrophils towards gradient sources of formyl-methionyl-leucyl-phenylalanine (FMLP) using 2 assay systems. The first resembled the micropore filter assay in that neutrophils on the upper surface of collagen gels were allowed to invade in the presence of either an isotropic concentration or a gradient of FMLP. Neutrophils invaded the gel vigorously in both cases. The effect of the gradient was assessed by determining the population distribution at different levels in the gel. Cells moving randomly should be distributed normally, and directional locomotion should cause deviation from normal distribution. Such a deviation was seen, but was of marginal significance. A more direct demonstration of chemotaxis was achieved by the second assay in which an agarose slab containing FMLP was incorporated into a gel, and the paths of nearby neutrophils were filmed. These cells showed an unequivocal directional response to the FMLP gradient. Protein gels can thus be used in the same way as both the presently used filter assays and visual assays using plane substrata, but with the advantage of providing a more physiological environment for the study of chemotaxis than either.