Ear Dermis Research Articles

Effects of Salivary Gland Homogenate from Wild‐Caught and Laboratory‐Reared Lutzomyia longipalpis on the Evolution and Immunomodulation of Leishmania (Leishmania) amazonensis Infection

We investigated the effects of Lutzomyia longipalpis salivary glands homogenate of wild-caught and laboratory-reared vectors on the lesion evolution and immunomodulation of the infection caused by Leishmania (Leishmania) amazonensis. To compare the effect of both salivary glands homogenate (SGH), C57BL/6 mice were inoculated subcutaneously into the hind footpads or into the ear dermis with 10(6) promastigotes in the presence or not of SGH from wild-caught and laboratory-colonized sand flies. Comparing SGH groups, the lesion size was lower in mice co-inoculated with wild-caught SGH, as the parasitism and the infiltration of macrophages at the inoculation site. Wild-caught SGH also determined lower production of IL-4 and IL-10 but higher IL-12 levels compared with laboratory-reared SGH. Our findings address a probable bias by using SGH from laboratory-colonized sand flies instead of wild-caught vector SGH in studies concerning saliva effects. A possible mild influence of sand fly saliva in natural infections caused by Leishmania is also speculated, as infection is transmitted by wild and not by laboratory-reared vectors.

Histopathology of Leishmania major infection: revisiting L. major histopathology in the ear dermis infection model

We describe the relationship between lesion outcome and histopathological hallmarks in susceptible (BALB/c) and resistant (C57BL/6 and IL-4-deficient BALB/c) mouse strains over the course of a 12-week-infection with Leishmania major in the ear. The infiltration of mononuclear cells and polymorphonuclear cells occurred within 6 h and mononuclear cells predominated one week post-infection. Permissive intracellular growth of the pathogen was associated with non-healing lesions. In contrast, tissue damage and clearance of the parasite was observed in healing lesions and was associated with inducible nitric oxide synthase expression. The identification of the structural components of tissue reaction to the parasite in this study furthers our understanding of subjacent immune effector mechanisms.

Despite Increased CD4+Foxp3+ Cells within the Infection Site, BALB/c IL-4 Receptor-Deficient Mice Reveal CD4+Foxp3-Negative T Cells as a Source of IL-10 inLeishmania majorSusceptibility

BALB/c IL-4Ralpha(-/-) mice, despite the absence of IL-4/IL-13 signaling and potent Th2 responses, remain highly susceptible to Leishmania major substain LV39 due exclusively to residual levels of IL-10. To address the contribution of CD4(+)CD25(+) T regulatory (Treg) cells to IL-10-mediated susceptibility, we depleted CD4(+)CD25(+) cells in vivo and reconstituted IL-4Ralpha x RAG2 recipients with purified CD4(+)CD25(-) T cells. Although anti-CD25 mAb treatment significantly decreased parasite numbers in IL-4Ralpha(-/-) mice, treatment with anti-IL-10R mAb virtually eliminated L. major parasites in both footpad and dermal infection sites. In addition, IL-4Ralpha x RAG2 mice reconstituted with CD4(+) cells depleted of CD25(+) Treg cells remained highly susceptible to infection. Analysis of L. major-infected BALB/c and IL-4Ralpha(-/-) inflammatory sites revealed that the majority of IL-10 was secreted by the CD4(+)Foxp3(-) population, with a fraction of IL-10 coming from CD4(+)Foxp3(+) Treg cells. All T cell IFN-gamma production was also derived from the CD4(+)Foxp3(-) population. Nevertheless, the IL-4Ralpha(-/-)-infected ear dermis, but not draining lymph nodes, consistently displayed 1.5- to 2-fold greater percentages of CD4(+)CD25(+) and CD4(+)Foxp3(+) Treg cells compared with the BALB/c-infected dermis. Thus, CD4(+)Foxp3(-) T cells are a major source of IL-10 that disrupts IFN-gamma activity in L. major-susceptible BALB/c mice. However, the increase in CD4(+)Foxp3(+) T cells within the IL-4Ralpha(-/-) dermis implies a possible IL-10-independent role for Treg cells within the infection site, and may indicate a novel immune escape mechanism used by L. major parasites in the absence of IL-4/IL-13 signaling.

Long-Term Efficacy of a Novel Ribose?Cross-linked Collagen Dermal Filler: A Histologic and Histomorphometric Study in an Animal Model

Degradation and loss of the three-dimensional shape are the major causes of limited functional longevity of dermal fillers made of natural polymers as collagen and hyaluronic acid. This study assessed the functional longevity of a new ribose-cross-linked collagen filler during 24 months in an animal model. Ribose-cross-linked collagen (Evolence, Colbar Life Sciences Ltd), glutaraldehyde-cross-linked collagen (Zyplast, Inamed Inc.), and non-cross-linked collagen (Zyderm, Inamed Inc.) were injected in the rabbit ear dermis. Biopsies obtained at 1, 6, 12, and 24 months were histomorphometrically assessed for shape preservation and cell repopulation. The three-dimensional shape of Evolence remained stable during 24 months. Zyderm and Zyplast lost their three-dimensional shape after 6 months. Although the cell density in Evolence remained stable over time, that in Zyplast and Zyderm decreased significantly at 12 and 24 months. Ribose-cross-linked collagen is endowed with a higher functional longevity as assessed in an animal model when compared with the most used collagen-based dermal fillers.

The prevention of the growth of Leishmania major progeny in BALB/c iron-loaded mice: a process coupled to increased oxidative burst, the amplitude and duration of which depend on initial parasite developmental stage and dose

BALB/c mice were given or not iron around the time of intradermal parasite inoculation, in their ears, of either 10 6 stationary-phase (designated “high-dose model”) or 10 3 Leishmania major metacyclic promastigotes (designated “low-dose model”). Iron-loaded mice in the high-dose model displayed delayed and limited pathogenic processes, whereas in the low-dose model, the mice remained ear lesion-free over 12 months post-parasite inoculation. These phenotypes were coupled to an increased leukocyte oxidative burst displayed mainly by neutrophils: it was early and transient in the high-dose model, whereas it was sustained in the low-dose model. In the latter model, injection of an antioxidant (diphenyleneiodonium chloride) at week 2 post- L. major inoculation resulted in a significant decrease in oxidative burst and reversed the protective status. The increased and sustained oxidative burst displayed by the neutrophils, the sustained presence of IL-12 (p40/p70)-positive leukocytes in the ear dermis, the low number of inflammatory leukocytes in the ear dermis and their concomitant high number in the draining lymph node are three related features that likely contribute to the shaping of the protective status, the onset and dynamic maintenance of which are antioxidant sensitive.

Genetic background influences immune responses and disease outcome of cutaneous L. mexicana infection in mice

The experimental model of high-dose Leishmania mexicana infection is used frequently to study molecular mechanisms regulating Th2 response since most inbred mice regardless of their genetic background display Th2 cytokine-dependent susceptibility to L. mexicana unlike Leishmania major. Here, we analyzed the course of L. mexicana infection in BALB/c, C57BL/6 and CBA/J mouse strains using low-dose ear infection model that mimics natural transmission. Although all three strains were equally susceptible to high-dose back rump L. mexicana infection, they displayed marked differences in their ability to control parasite growth after low-dose ear infection. Leishmania mexicana-infected BALB/c mice produced high levels of Th2-associated cytokines and developed non-healing lesions full of parasites, whereas CBA/J mice preferentially produced Th1-associated IFN-gamma but low levels of IL-4, and developed small self-resolving lesions. Both BALB/c and C57BL/6 mice produced comparable amounts of IFN-gamma following L. mexicana infection, but later produced less Th2-associated cytokines, and exhibited an 'intermediate' susceptibility phenotype characterized by lesion sizes that were significantly smaller than BALB/c mice but larger than CBA/J mice. Interestingly, all three strains also showed marked differences in trafficking of macrophages, CD4+ T cells and CD8+ T cells into their lesions. Finally, we analyzed the course of low-dose L. mexicana infection in signal transducers and activators of transcription (STAT) 6-/- and STAT6+/+ BALB/c mice. We found that STAT6-/- mice mount a Th1 response, produce high levels of IL-12 and IFN-gamma and develop smaller lesions containing fewer parasites as compared with STAT6+/+ mice. Our findings demonstrate that genetic background plays a critical role in determining susceptibility of inbred mice to low-dose L. mexicana infection. Furthermore, together with our previous findings, they show that STAT6-mediated signaling is involved in mediating susceptibility to L. mexicana following both high-dose back rump and low-dose ear dermis infection.

Neutrophils rapidly migrate via lymphatics after Mycobacterium bovis BCG intradermal vaccination and shuttle live bacilli to the draining lymph nodes

AbstractThe early innate response after Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccination is poorly characterized but probably decisive for subsequent protective immunity against tuberculosis. Therefore, we vaccinated mice with fluorescent BCG strains in the ear dorsum, as a surrogate of intradermal vaccination in humans. During the first 3 days, we tracked BCG host cells migrating out of the dermis to the auricular draining lymph nodes (ADLNs). Resident skin dendritic cells (DCs) or macrophages did not play a predominant role in early BCG capture and transport to ADLNs. The main BCG host cells rapidly recruited both in the dermis and ADLNs were neutrophils. Fluorescent green or red BCG strains injected into nonoverlapping sites were essentially sheltered by distinct neutrophils in the ADLN capsule, indicating that neutrophils had captured bacilli in peripheral tissue and transported them to the lymphoid organ. Strikingly, we observed BCG-infected neutrophils in the lumen of lymphatic vessels by confocal microscopy on ear dermis. Fluorescence-labeled neutrophils injected into the ears accumulated exclusively into the ipsilateral ADLN capsule after BCG vaccination. Thus, we provide in vivo evidence that neutrophils, like DCs or inflammatory monocytes, migrate via afferent lymphatics to lymphoid tissue and can shuttle live microorganisms. (Blood. 2005;106: 1843-1850)

Toward a Novel Experimental Model of Infection To Study American Cutaneous Leishmaniasis Caused byLeishmania braziliensis

Leishmania spp. cause a broad spectrum of diseases collectively known as leishmaniasis. Leishmania braziliensis is the main etiological agent of American cutaneous leishmaniasis (ACL) and mucocutaneous leishmaniasis. In the present study, we have developed an experimental model of infection that closely resembles ACL caused by L. braziliensis. In order to do so, BALB/c mice were infected in the ear dermis with 10(5) parasites and distinct aspects of the infection were evaluated. Following inoculation, parasite expansion in the ear dermis was accompanied by the development of an ulcerated dermal lesion which healed spontaneously, as seen by the presence of a scar. Histological analysis of infected ears showed the presence of a mixed inflammatory infiltrate consisting of both mononuclear and polymorphonuclear cells. In draining lymph nodes, parasite replication was detected throughout the infection. In vitro restimulation of draining lymph node cells followed by intracellular staining showed an up-regulation in the production of gamma interferon (IFN-gamma) and in the frequency of IFN-gamma-secreting CD4(+) and CD8(+) T cells. Reverse transcription-PCR of ears and draining lymph node cells showed the expression of CC chemokines. The dermal model of infection with L. braziliensis herein is able to reproduce aspects of the natural infection, such as the presence of an ulcerated lesion, parasite dissemination to lymphoid areas, and the development of a Th1-type immune response. These results indicate that this model shall be useful to address questions related to the concomitant immunity to reinfection and parasite persistence leading to mucocutaneous leishmaniasis.

Conditions Influencing the Efficacy of Vaccination with Live Organisms againstLeishmania majorInfection

Numerous experimental vaccines have been developed with the goal of generating long-term cell-mediated immunity to the obligate intracellular parasite Leishmania major, yet inoculation with live, wild-type L. major remains the only successful vaccine in humans. We examined the expression of immunity at the site of secondary, low-dose challenge in the ear dermis to determine the kinetics of parasite clearance and the early events associated with the protection conferred by vaccination with live L. major organisms in C57BL/6 mice. Particular attention was given to the route of vaccination. We observed that the rapidity, strength, and durability of the memory response following subcutaneous vaccination with live parasites in the footpad are even greater than previously appreciated. Antigen-specific gamma interferon (IFN-gamma)-producing T cells infiltrate the secondary site by 1.5 weeks, and viable parasites are cleared as early as 2.5 weeks following rechallenge, followed by a rapid drop in IFN-gamma(+) CD4(+) cell numbers in the site. In comparison, intradermal vaccination with live parasites in the ear generates immunity that is delayed in effector cell recruitment to the rechallenge site and in the clearance of parasites from the site. This compromised immunity was associated with a rapid recruitment of interleukin-10 (IL-10)-producing CD4(+) T cells to the rechallenge site. Treatment with anti-IL-10-receptor or anti-CD25 antibody enhanced early parasite clearance in ear-vaccinated mice, indicating that chronic infection in the skin generates a population of regulatory cells capable of influencing the level of resistance to reinfection. A delicate balance of effector and regulatory T cells may be required to optimize the potency and durability of vaccines against Leishmaniasis and other intracellular pathogens.

Tissue‐selective mast cell reconstitution and differential lung gene expression in mast cell‐deficient KitW‐sh/KitW‐sh sash mice

Mast cell-deficient Kit(W)/Kit(W-v) mice are an important resource for studying mast cell functions in vivo. However, because they are compound heterozygotes in a mixed genetic background and are infertile, they cannot be crossed easily with other mice. To overcome this limitation, we explored the use of Kit(W-sh)/Kit(W-sh) mice for studying mast cell biology in vivo. These mice are in a C57BL/6 background, are fertile and can be bred directly with other genetically modified mice. Ten-week-old Kit(W-sh)/Kit(W-sh) are profoundly mast cell-deficient. No mast cells are detected in any major organ, including the lung. Gene microarrays detect differential expression of just seven of 16,463 genes in lungs of Kit(W-sh)/Kit(W-sh) mice compared with wild-type mice, indicating that resting mast cells regulate expression of a small set of genes in the normal lung. Injecting 10(7) bone marrow-derived mast cells (BMMC) into tail veins of Kit(W-sh)/Kit(W-sh) mice reconstitutes mast cell populations in lung, stomach, liver, inguinal lymph nodes, and spleen, but not in the tongue, trachea or skin. Injection of BMMC into ear dermis or peritoneum reconstitutes mast cells locally in these tissues. When splenectomized Kit(W-sh)/Kit(W-sh) mice are intravenously injected with BMMC, mast cells circulate longer and are found more often in the liver and inguinal lymph nodes, indicating that the spleen acts as a reservoir for mast cells following injection and limits migration to some tissues. In summary, these findings show that mast cell-deficient Kit(W-sh)/Kit(W-sh) mice possess unique attributes that favour their use for studying mast cell functions in vivo.

Bioluminescent Leishmania expressing luciferase for rapid and high throughput screening of drugs acting on amastigote-harbouring macrophages and for quantitative real-time monitoring of parasitism features in living mice

In this study, we have established conditions for generating Leishmania amazonensis recombinants stably expressing the firefly luciferase gene. These parasites produced significant bioluminescent signals for both in vitro studies and the development of an in vivo model, allowing the course of the parasitism to be readily monitored in real time in the living animals such as laboratory mice. First, a model was established, using parasite-infected mouse macrophages for rapidly determining the activity of drugs against intracellular amastigotes. Results indicated that recombinant Leishmania can be reliably and confidently used to monitor compounds acting on intracellular amastigote-harbouring macrophages. Secondly, temporal analyses were performed following inoculation of metacyclic promastigotes into the ear dermis of BALB/c mice and the bioluminescent light transmitted through the tissue was imaged externally using a charge coupled device (CCD) camera. Bioluminescent signals, measured at the inoculation site and in the draining lymph node of mice containing these parasites correlated well with the more classical quantification of parasites. These assays prove that the real-time bioluminescent assay is not only sensitive but also more rapid than culture-base techniques allowing to monitor parasite-load before any clinical signs of leishmaniasis are detectable. In short, this luciferase imaging study is useful to monitor the efficacy of anti-leishmanial drugs on live cell culture and to trace leishmanial infection in animal models.

Intradermal inoculations of low doses of Leishmania major and Leishmania amazonensis metacyclic promastigotes induce different immunoparasitic processes and status of protection in BALB/c mice

In order to simulate the natural long term parasitisms which may occur in mammals infected with Leishmania, cutaneous leishmaniases due to Leishmania major or Leishmania amazonensis were induced using a model based on the inoculation of 10–1000 metacyclic promastigotes into the ear dermis of BALB/c mice. The final outcome of these parasitisms was dependent upon the number of inoculated parasites. Only some of the mice inoculated with ten parasites displayed cutaneous lesions, whereas most mice infected with 100 metacyclics and all mice infected with 1000 metacyclics developed progressive lesions. We found, using the latter experimental conditions, that the onset of the pathology was associated with: (a) parasite multiplication in the inoculation site and the draining lymph node correlating with an increase of the lymph node cell number, especially in L. major-infected mice; and (b) the detection of lymph node cells, at least in part CD4 + T lymphocytes, able to produce high levels of interferon-γ, interleukin (IL)-4, IL-10 and IL-13. Thereafter, mice infected by L. major harboured few parasites in the ear and had a 100-fold reduction in lymph node parasite load between 23 and 40 weeks post-inoculation. In contrast, the parasite loads of L. amazonensis-infected mice remained stable in the ear and increased in nodes during the same period of time. Only L. major-infected mice that exhibited cutaneous lesions in the primary site were resistant to the re-inoculation of 1000 metacyclic promastigotes, whereas all L. amazonensis-primary infected mice remained susceptible to a second homologous challenge. These results are the first to document that a status of resistance to re-infection, referred to concomitant immunity, is coupled to the development of primary progressive lesions in L. major-infected BALB/c mice. Such a protective status is absent in L. amazonensis-infected BALB/c mice.

The levels and patterns of cytokines produced by CD4 T lymphocytes of BALB/c mice infected with Leishmania major by inoculation into the ear dermis depend on the infectiousness and size of the inoculum.

The production of cytokines by CD4 lymph node T lymphocytes derived from BALB/c mice recently infected in the ear dermis with high (10(6) parasites) or low (10(3) parasites) doses of Leishmania major metacyclic promastigotes (MP) was examined over a 3-week period following inoculation. Results were compared with those obtained when mice were injected with less infectious parasite populations, namely, stationary-phase or log-phase promastigotes (LP). Cells were purified 16 h and 3, 8, and 19 days after inoculation, and the amounts of gamma interferon (IFN-gamma) and interleukin-4 (IL-4) released in response to LACK (Leishmania homolog of receptors for activated C kinase) or total L. major antigens were assessed. We found that LACK-reactive T cells from mice inoculated with a high dose of parasites first produced IFN-gamma and later on IL-4; the level of IFN-gamma produced early by these cells was dependent upon the stage of the promastigotes inoculated, the highest level being reached with cells recovered from mice inoculated with the least infectious parasites, LP; sequential production of IFN-gamma and then of IL-4 also characterized L. major antigen-reactive CD4 T cells, suggesting that the early production of IFN-gamma does not impede the subsequent rise of IL-4 and finally the expansion of the parasites; after low-dose inoculation of MP, cutaneous lesions developed with kinetics similar to that of lesions induced after inoculation of 10(6) LP, but in this case CD4 T lymphocytes did not release IFN-gamma or IL-4 in the presence of LACK and neither cytokine was produced in response to L. major antigens before the onset of lesion signs. These results suggest the existence of a discreet phase in terms of CD4 T-cell reactivity for at least the first 8 days following inoculation, a time period during which parasites are able to grow moderately. In conclusion, the levels and profiles of cytokines produced by Leishmania-specific CD4 T lymphocytes clearly depend on both the stage of differentiation and number of parasites used for inoculation.

CD8+ T cells are required for primary immunity in C57BL/6 mice following low-dose, intradermal challenge with Leishmania major.

Standard murine models of cutaneous leishmaniasis, involving s.c. inoculation of large numbers of Leishmania major promastigotes, have not supported an essential role for CD8(+) T cells in the control of primary infection. Recently, a L. major model combining two main features of natural transmission, low parasite dose and inoculation into a dermal site, has been established in resistant C57BL/6 mice. In the present studies, C57BL/6 mice with CD8(+) T cell deficiencies, including CD8(-/-) and CD8-depleted mice, failed to control the growth of L. major following inoculation of 100 metacyclic promastigotes into the ear dermis. The resulting dermal pathology was minor and delayed. Lesion formation in wild-type mice was coincident with the killing of parasites in the inoculation site. Both events were associated with the accumulation of CD8(+) T lymphocytes in the skin and with the capacity of CD8(+) T cells recovered from draining lymph nodes or infected dermis to release IFN-gamma following coculture with infected dendritic cells. Reconstitution of resistance to L. major in RAG(-/-) mice using T cells from naive donors was optimal when both CD4(+) and CD8(+) T cells were transferred. Primed CD8(+) T lymphocytes obtained from C57BL/6 mice during the acute stage of infection were able to mediate both pathology and immunity when transferred alone. The low dose, intradermal challenge model reveals that CD8(+) T cells play an essential role in both pathogenesis of and immunity to primary infection with L. major in the skin.

Leishmania major reaches distant cutaneous sites where it persists transiently while persisting durably in the primary dermal site and its draining lymph node: a study with laboratory mice.

So far, studies of Leishmania persistence in mice have used injections of parasites administered either intravenously in the tail vein or subcutaneously in the footpad. These routes poorly reflect the natural conditions when the sandfly delivers metacyclic promastigotes intradermally. In this study B10D2 and BALB/c mice were inoculated within the ear dermis with 10(4) Leishmania major metacyclic promastigotes. The parasite load was monitored by quantitative PCR in different tissues from the dermal inoculation site to distant tissues. The two sites of multiplication and persistence of parasites were the site of L. major inoculation and the draining lymph node (DLN), with a different pattern in the two mouse inbred lines. These two organs were the only sites harboring parasites 12 months postinoculation, with the DLN of BALB/c mice harboring around 10(7) parasites, a stable load from months 3 to 12. In these two sites, 8 and 12 months after inoculation, interleukin 4 (IL-4), gamma interferon, and inducible nitric oxide synthase transcripts parallel the parasite load while IL-10 transcript levels remain high. In addition, at early time points until month 3, parasite DNA was also detected in distant tissues such as the contralateral noninoculated ear or the tail skin, indicating that blood was at least transiently disseminating the parasites. In contrast, L. major DNA in liver, spleen, and femoral bone marrow remained sporadic in mice of both lines. This study is discussed within the framework of Leishmania transmission from the vertebrate host to the sandfly vector, a complex process still poorly understood.

Transient IFN-gamma synthesis in the lymph node draining a dermal site loaded with UV-irradiated herpes simplex virus type 1: an NK- and CD3-dependent process regulated by IL-12 but not by IFN-alpha/beta.

Our previous studies have shown that UV-inactivated, non-replicating herpes simplex virus type 1 (UV-HSV-1) triggers early and transient synthesis of IFN-alpha/beta in the mouse regional lymph node when delivered upstream (i.e. in the ear dermis). In this study, it is demonstrated, by use of a quantitative RT-PCR readout assay, that IFN-gamma mRNA expression was rapidly and transiently upregulated in draining lymph nodes when UV-HSV-1 was delivered in the ear dermis of C57Bl/6 mice. An increased number of IFN-gamma-producing cells was also detected in the lymph node by flow cytometric analysis. Two different subsets of cells, namely DX5(+) NK cells and CD3epsilon(+) T cells, accounted for this early IFN-gamma synthesis. Prompt upregulation of IFN-alpha and IL-12p40 mRNA was also recorded. We took advantage of IFN-alpha/beta-receptor knockout and wild-type 129 mice to study a potential role of IFN-alpha/beta in the signalling pathway leading to IFN-gamma transcription/translation. IFN-gamma mRNA upregulation still occurred in IFN-alpha/beta-receptor(-/-) mice, showing that IFN-alpha/beta was dispensable. The use of IL-12-neutralizing antibodies, prior to UV-HSV-1 delivery, confirmed the major role played by IL-12 in the early/transient IFN-gamma burst.

Development of a natural model of cutaneous leishmaniasis: powerful effects of vector saliva and saliva preexposure on the long-term outcome of Leishmania major infection in the mouse ear dermis.

We have developed a model of cutaneous leishmaniasis due to Leishmania major that seeks to mimic the natural conditions of infection. 1,000 metacyclic promastigotes were coinoculated with a salivary gland sonicate (SGS) obtained from a natural vector, Phlebotomus papatasii, into the ear dermis of naive mice or of mice preexposed to SGS. The studies reveal a dramatic exacerbating effect of SGS on lesion development in the dermal site, and a complete abrogation of this effect in mice preexposed to salivary components. In both BALB/c and C57Bl/6 (B/6) mice, the dermal lesions appeared earlier, were more destructive, and contained greater numbers of parasites after infection in the presence of SGS. Furthermore, coinoculation of SGS converted B/6 mice into a nonhealing phenotype. No effect of SGS was seen in either IL-4- deficient or in SCID mice. Disease exacerbation in both BALB/c and B/6 mice was associated with an early (6 h) increase in the frequency of epidermal cells producing type 2 cytokines. SGS did not elicit type 2 cytokines in the epidermis of mice previously injected with SGS. These mice made antisaliva antibodies that were able to neutralize the ability of SGS to enhance infection and to elicit IL-4 and IL-5 responses in the epidermis. These results are the first to suggest that for individuals at risk of vector-borne infections, history of exposure to vector saliva might influence the outcome of exposure to transmitted parasites.

Comparative Study of Survival of Autologous Adipose Tissue Taken and Transplanted by Different Techniques

In recent years, adipocytes obtained by suction-assisted lipectomy have been used for implantation by injection methods. This study is designed to assess the appearance of suctioned and excised adipose tissue and its survival after being injected or implanted into different tissues (0.5 cc into the rectus muscle and 0.5 cc into the dorsal ear skin) of New Zealand White rabbits. The results showed that significant numbers of adipocytes were ruptured after suction procedures. The intact cells represented approximately 10 percent of the fat cell population. Fat cells in aspirated and excised samples remained intact and did not differ histologically. After being injected into tissue, adipocytes appeared to survive better for a short term in a more vascularized bed (rectus muscle) than in a low vascular area (ear dermis). Long-term studies at 6- to 9-month intervals revealed transplanted adipose tissue, taken by suction or excision, being replaced with fibrosis, although cystic spaces and only a small number of surviving adipocytes were still present. Insulin did not show any protective effects on survival of the adipocytes during their transplantation.

The keratin polypeptide patterns in heterotypically recombined epithelia of skin and mucosa of adult mouse

Epithelial-mesenchymal interactions were investigated considering both morphologic criteria and keratin polypeptide expression in homotypic and heterotypic recombinants of adult mouse skin and oral mucosa. Two series of cross-recombinants of epithelia with different morphology and keratin patterns were chosen: (a) footpad epidermis/ear dermis and ear epidermis/footpad dermis; (b) palate epithelium/cheek connective tissue and cheek epithelium/palate connective tissue. Homotypic and heterotypic recombinants were prepared after EDTA-separation of the original tissues and then grown on syngeneic mice in subcutaneously prepared protected graft chambers. EDTA-separation is especially suited to completely separate the epidermal-dermal union, and the transplantation procedure used strictly prevents contamination with host epithelium. Five weeks after implantation keratins were analyzed by one and two-dimensional gel electrophoresis and peptide mapping. In both series, homotypic recombination of the tissues did not alter the original morphology and keratin polypeptide composition of the individual epithelial components. Ear epidermis displayed no significant changes in structure or keratin pattern in heterotypic recombinants. Recombined with ear dermis, footpad epidermis showed acquisition of some morphologic features typical for ear epidermis and slight changes in keratin composition which were, however, difficult to interpret due to the normal similarities of footpad keratin with that of ear. In contrast, the hetero-recombinants of the palate/cheek series exhibited considerable alterations in their keratin patterns. Either epithelium showed suppression of distinct keratin subunits and de novo expression of subunits characteristic of the epithelium normally associated with the connective tissue component. The keratin patterns of both matches closely resembled each other and represented patterns intermediate between the normal patterns. This partial, however, significant modulation in the expression of differentiation markers was paralleled by similarly directed changes in the architecture of the heterotransplanted tissues, thus indicating that both morphogenesis and cytodifferentiation of certain adult epithelia can be influenced by extrinsic mesenchymal factors.

Vibrissa dermal papilla cell aggregative behaviour in vivo and in vitro

Parallel cultures of adult rat vibrissa dermal papilla cells and skin fibroblasts revealed differences between the two cell types with respect to a number of criteria. In particular the dermal papilla cells demonstrated a distinctive single cell morphology, and at confluence formed cell aggregates radically different from regular fibroblast multilayering and patterning. This finding confirmed repeated observations of papilla cell clumping in short-term culture. The dermal papilla cells which are mitotically quiescent in situ were also shown to have a lower proliferative capacity than the skin fibroblasts. The affinity shown by papilla cells towards each other in culture reflected the behaviour demonstrated by isolated dermal papillae transplanted into ear dermis and into the collagenous capsule of the vibrissa follicle. In the absence of epidermal contact the papilla cells remained as recognizable rounded aggregates for the experimental period of up to nine months. Synthesis of extracellular material typical of that seen in situ was observed, particularly during the first weeks following transplantation. The collective behaviour of the dermal papilla cells revealed in this study may be significant for the morphogenetic activity of the papilla, and for papilla size during the hair cycle. It may also reflect the retention of embryonic-like properties by the dermal component of adult hair follicles.