Application of a recombinase polymerase amplification (RPA) assay and pilot field testing for Giardia duodenalis at Lake Albert, Uganda.

BACKGROUND: Giardia duodenalis is a gastrointestinal protozoan causing 184 million cases of giardiasis worldwide annually. Detection is by microscopy or coproantigen assays, although sensitivity is often compromised by intermittent shedding of cysts or trophozoites, or operator expertise. Therefore, for enhanced surveillance field-applicable, point-of-care (POC), molecular assays are needed. Our aims were to: (i) optimise the recombinase polymerase amplification (RPA) assay for the isothermal amplification of the G. duodenalis ß-giardin gene from trophozoites and cysts, using published primer and probes; and (ii) perform a pilot field validation of RPA at a field station in a resource-poor setting, on DNA extracted from stool samples from schoolchildren in villages around Lake Albert, Uganda. Results were compared to an established laboratory small subunit ribosomal RNA (SSU rDNA) qPCR assay with additional testing using a qPCR targeting the triose phosphate isomerase (tpi) DNA regions that can distinguish G. duodenalis of two different assemblages (A and B), which are human-specific. RESULTS: Initial optimisation resulted in the successful amplification of predicted RPA products from G. duodenalis-purified gDNA, producing a double-labelled amplicon detected using lateral flow strips. In the field setting, of 129 stool samples, 49 (37.9%) were positive using the Giardia/Cryptosporidium QuikChek coproantigen test; however, the RPA assay when conducted in the field was positive for a single stool sample. Subsequent molecular screening in the laboratory on a subset (n = 73) of the samples demonstrated better results with 21 (28.8%) RPA positive. The SSU rDNA qPCR assay resulted in 30/129 (23.3%) positive samples; 18 out of 73 (24.7%) were assemblage typed (9 assemblage A; 5 assemblage B; and 4 mixed A+B). Compared with the SSU rDNA qPCR, QuikChek was more sensitive than RPA (85.7 vs 61.9%), but with similar specificities (80.8 vs 84.6%). In comparison to QuikChek, RPA had 46.4% sensitivity and 82.2% specificity. CONCLUSIONS: To the best of our knowledge, this is the first in-field and comparative laboratory validation of RPA for giardiasis in low resource settings. Further refinement and technology transfer, specifically in relation to stool sample preparation, will be needed to implement this assay in the field, which could assist better detection of asymptomatic Giardia infections.

Refining wet lab experiments with in silico searches: A rational quest for diagnostic peptides in visceral leishmaniasis.

BACKGROUND: The search for diagnostic biomarkers has been profiting from a growing number of high quality sequenced genomes and freely available bioinformatic tools. These can be combined with wet lab experiments for a rational search. Improved, point-of-care diagnostic tests for visceral leishmaniasis (VL), early case detection and surveillance are required. Previous investigations demonstrated the potential of IgG1 as a biomarker for monitoring clinical status in rapid diagnostic tests (RDTs), although using a crude lysate antigen (CLA) as capturing antigen. Replacing the CLA by specific antigens would lead to more robust RDTs. METHODOLOGY: Immunoblots revealed L. donovani protein bands detected by IgG1 from VL patients. Upon confident identification of these antigens by mass spectrometry (MS), we searched for evidence of constitutive protein expression and presence of antigenic domains or high accessibility to B-cells. Selected candidates had their linear epitopes mapped with in silico algorithms. Multiple high-scoring predicted epitopes from the shortlisted proteins were screened in peptide arrays. The most promising candidate was tested in RDT prototypes using VL and nonendemic healthy control (NEHC) patient sera. RESULTS: Over 90% of the proteins identified from the immunoblots did not satisfy the selection criteria and were excluded from the downstream epitope mapping. Screening of predicted epitope peptides from the shortlisted proteins identified the most reactive, for which the sensitivity for IgG1 was 84% (95% CI 60-97%) with Sudanese VL sera on RDT prototypes. None of the sera from NEHCs were positive. CONCLUSION: We employed in silico searches to reduce drastically the output of wet lab experiments, focusing on promising candidates containing selected protein features. By predicting epitopes in silico we screened a large number of peptides using arrays, identifying the most promising one, for which IgG1 sensitivity and specificity, with limited sample size, supported this proof of concept strategy for diagnostics discovery, which can be applied to the development of more robust IgG1 RDTs for monitoring clinical status in VL.

Synthetic peptides as a novel approach for detecting antibodies against sand fly saliva.

BACKGROUND: Hosts repeatedly bitten by sand flies develop antibodies against sand fly saliva and screening of these immunoglobulins can be employed to estimate the risk of Leishmania transmission, to indicate the feeding preferences of sand flies, or to evaluate the effectiveness of vector control campaigns. Previously, antibodies to sand fly saliva were detected using whole salivary gland homogenate (SGH) or recombinant proteins, both of which also have their disadvantages. This is the first study on sand flies where short peptides designed based on salivary antigens were successfully utilized for antibody screening. METHODOLOGY/PRINCIPAL FINDINGS: Specific IgG was studied in hosts naturally exposed to Phlebotomus orientalis, the main vector of Leishmania donovani in East Africa. Four peptides were designed by the commercial program EpiQuest-B, based on the sequences of the two most promising salivary antigens, yellow-related protein and ParSP25-like protein. Short amino acid peptides were synthesised and modified for ELISA experiments. Specific anti-P. orientalis IgG was detected in sera of dogs, goats, and sheep from Ethiopia. The peptide OR24 P2 was shown to be suitable for antibody screening; it correlated positively with SGH and its specificity and sensitivity were comparable or even better than that of previously published recombinant proteins. CONCLUSIONS/SIGNIFICANCE: OR24 P2, the peptide based on salivary antigen of P. orientalis, was shown to be a valuable tool for antibody screening of domestic animals naturally exposed to P. orientalis. We suggest the application of this promising methodology using species-specific short peptides to other sand fly-host combinations.

Association of Diet With Skin Histological Features in UV-B-Exposed Mice.

IMPORTANCE: Long-term exposure to solar radiation produces deleterious photoaging of the skin. It is not known if diet can influence skin photoaging. OBJECTIVES: To study the influence of a calorie-restricted diet and an obesity diet in mice exposed to long-term UV-B irradiation to assess if there is an association between diet and histopathological response to UV-B irradiation. DESIGN, SETTING, AND PARTICIPANTS: In this animal model study in an academic setting, the dorsal skin of SKH1 hairless mice receiving normal, calorie-restricted, and obesity diets was exposed to UV-B irradiation 3 times a week for 10 weeks and were compared with corresponding controls. The mice were placed in the following groups, with 8 animals in each group: (1) intact control (C) with regular diet and no UV-B exposure, (2) intact control with UV-B exposure (CR), (3) calorie-restricted diet (CrC), (4) calorie-restricted diet with UV-B exposure (CrR), (5) obesity diet (OC), and (6) obesity diet with UV-B exposure (OR). The experiment was conducted during October through December 2013. Tissue processing and histological analysis were completed in 2016. MAIN OUTCOMES AND MEASURES: Histomorphometric analysis was performed on paraffin-embedded skin sections stained by histological and immunohistochemical methods for estimation of epidermal thickness, epidermal proliferating cell nuclear antigen index, collagen I, elastic fibers, fibroblasts, mast cells, dermal cellularity, and adipose layer ratio. Changes in wrinkles were noted. RESULTS: Hairless female mice (age range, 6-8 weeks) were obtained. With a normal diet, changes from UV-B irradiation occurred in epidermal thickness, epidermal proliferating cell nuclear antigen index, collagen I, elastic fibers, fibroblasts, and mast cells, which were modestly influenced by an obesity diet. Calorie restriction influenced the skin in nonirradiated control animals, with higher values for most variables. After UV-B exposure in animals with calorie restriction, epidermal thickness was increased, but other variables were unaffected. Animals receiving the calorie-restricted diet lost weight when exposed to long-term UV-B irradiation. Wrinkles were reduced in the calorie-restricted control group and in UV-B-exposed animals who received the obesity diet. CONCLUSIONS AND RELEVANCE: Dietary alterations seem to modify histopathological responses to UV-B exposure in the skin of hairless mice. LEVEL OF EVIDENCE: NA.

Development of peptide-based lineage-specific serology for chronic Chagas disease: geographical and clinical distribution of epitope recognition.

BACKGROUND: Chagas disease, caused by infection with the protozoan Trypanosoma cruzi, remains a serious public health issue in Latin America. Genetically diverse, the species is sub-divided into six lineages, known as TcI-TcVI, which have disparate geographical and ecological distributions. TcII, TcV, and TcVI are associated with severe human disease in the Southern Cone countries, whereas TcI is associated with cardiomyopathy north of the Amazon. T. cruzi persists as a chronic infection, with cardiac and/or gastrointestinal symptoms developing years or decades after initial infection. Identifying an individual's history of T. cruzi lineage infection directly by genotyping of the parasite is complicated by the low parasitaemia and sequestration in the host tissues. METHODOLOGY/PRINCIPAL FINDINGS: We have applied here serology against lineage-specific epitopes of the T. cruzi surface antigen TSSA, as an indirect approach to allow identification of infecting lineage. Chagasic sera from chronic patients from a range of endemic countries were tested by ELISA against synthetic peptides representing lineage-specific TSSA epitopes bound to avidin-coated ELISA plates via a biotin labelled polyethylene glycol-glycine spacer to increase rotation and ensure each amino acid side chain could freely interact with their antibodies. 79/113 (70%) of samples from Brazil, Bolivia, and Argentina recognised the TSSA epitope common to lineages TcII/TcV/TcVI. Comparison with clinical information showed that a higher proportion of Brazilian TSSApep-II/V/VI responders had ECG abnormalities than non-responders (38% vs 17%; p<0.0001). Among northern chagasic sera 4/20 (20%) from Ecuador reacted with this peptide; 1/12 Venezuelan and 1/34 Colombian samples reacted with TSSApep-IV. In addition, a proposed TcI-specific epitope, described elsewhere, was demonstrated here to be highly conserved across lineages and therefore not applicable to lineage-specific serology. CONCLUSIONS/SIGNIFICANCE: These results demonstrate the considerable potential for synthetic peptide serology to investigate the infection history of individuals, geographical and clinical associations of T. cruzi lineages.

Cosmeceutical effect on skin surface profiles and epidermis in UV-B-irradiated mice.

IMPORTANCE: These data may be useful for developing guidelines for clinicians and the general population related to the reversal of photoaging effects on the aging face damaged by solar radiation. OBJECTIVE: To investigate antiaging effects of 4 commercially available topical agents on the dorsal skin in photoaged hairless mice. DESIGN AND SETTING: Animal study at an academic medical center. Animals comprised 56 female Skh-1 hairless mice (6-8 weeks old). Skin samples were collected from nonirradiated intact mice (control), mice irradiated with UV-B for 8 weeks, mice irradiated with UV-B and then exposed to a topical cosmeceutical applied for 5 weeks, and UV-B-irradiated mice not exposed to cosmeceuticals and retained for 5 weeks until the end of the experiment. INTERVENTION: The mice were exposed to UV-B light 3 times a week for 2 months, followed by topical application of a peptide, antioxidant, estrogen, and retinoic acid agent for 5 weeks. MAIN OUTCOMES AND MEASURES: Surface features such as wrinkling were analyzed from replicas along with histomorphometric determination of epidermal thickness, sebocyte counts, and immunohistochemical study of proliferating cell nuclear antigen (PCNA). RESULTS: Exposure to UV-B induced significant wrinkle formation after 13 weeks, which was attenuated with treatments with a peptide cream, antioxidant mixture, and estrogen cream (mean [SD] Rz values: control [C], 60.7 [19.0]; irradiated [RAD], 51.8 [15.9] [P < .001]; irradiated-long [RAD-long], 86.0 [28.3] [P = .01]; antioxidant [AO], 45.2 [13.2]; peptide, 63.4 [18.8], estrogen, 64.6 [21.2]; retinoic acid [RA], 73.9 [28.5]; RAD-long vs C [P = .01], vs RAD [P < .001], vs estrogen [P = .04], vs peptide [P = .02], vs AO [P<.001], vs RA [P = .25]. There was a trend of reversal of irradiation-induced augmentation of epidermal thickness in animals treated with the peptide and AO (mean [SD] epidermal width: C, 21.0 [2.2] µm; RAD, 41.3 [7.0] µm [P < .001]; RAD-long, 39.1 [11.0] µm [P = .006]; AO, 37.3 [14] µm [P < .001]; peptide, 33.9 [3.8] µm [P = .01]; estrogen, 59.2 [9.2] µm [P = .003]; RA, 52.4 [8.7] µm [P < .001]). Retinoic acid augmented epidermal width and sebocyte counts (mean [SD] sebocyte data [number per gland]: C, 9.4 [2.0]; RAD, 11.69 [1.5] [P < .001]; RAD-long, 6.5 [1.3] [P = .73]; peptide, 7.2 [1.7] [P = .03]; estrogen, 4.1 [0.9] [P < .001]; AO, 7.2 [1.7] [P = .06]; RA, 11.0 [1.4] [P = .01]). Estrogen cream was effective in restoring surface features but enhanced thickness of epidermis in irradiated specimens. All groups had a higher PCNA index score except for peptide treatment, which brought it down to the control level (mean [SD] PCNA index values: C, 17.3 [1.5]; RAD, 32.4 [6.8] [P < .001]; RAD-long, 34.0 [6.1] [P < .001]; AO, 62.1 [3.5] [P = .01]; peptide, 20.1 [6.3] [P < .001]; estrogen, 56.8 [10.0] [P < .001]; RA, 35.2 [10.2] [P < .001]). CONCLUSIONS AND RELEVANCE: Of the 4 cosmeceuticals tested within this experimental period, peptide cream and antioxidant mixture were the most effective overall in reversing photoaging effects; retinoic acid was the least effective of these topical agents. LEVEL OF EVIDENCE: NA.

Effects of topicals on the aging skin process.

This article summarizes the antiaging properties of retinoids, glycolic acid, ascorbic acid, and peptide topicals. The supporting evidence is taken from the literature and the primary author's research, consisting of previously published data and new results from ongoing projects.

Epidermal cell proliferation in calorie-restricted aging rats.

Calorie restriction (CR) has been known to produce many beneficial health effects, and lowered cell proliferation from CR has been shown to produce anti-cancer effects in some tissues. In this study the rate of epidermal cell proliferation in aging Fischer 344 rats from ad libitum fed (AL) and CR colonies was assessed in relation to changes in epidermal thickness with age. Proliferating cell nuclear antigen (PCNA) was detected using immunohistochemical method on paraffin sections in the epidermis of dorsal skin and footpad in these animals obtained from the National Institute on Aging. The proliferating cell index was compared with morphometric measurement of epidermis in young, young adult and old animals (six per group). Data were analyzed by Excel and SPSS 14.0 softwares for statistical evaluation. Two-way analysis of variance (ANOVA) was applied to data to test the effects of age, diet, and age-diet interaction. The following significant effects were noted: (I) age and age-diet effects in dorsal skin epidermal width, and PCNA; (II) age, and diet effect on footpad epidermal thickness, and PCNA index. There was a trend of increasing epidermal thickness in the dorsal skin in normally feeding aging rats which was depressed with CR in the two younger groups. PCNA index showed a trend of attrition from young to old. The thickness of epidermis in foot pad showed a curvilinear trend in both AL and CR groups with lowest mean values in the old group, and more predominant effect in CR-exposed animals. The proliferation index in the foot pad demonstrated a trend of reduction in old specimens with lower mean values in each corresponding CR age group. This report agrees with CR-inhibited cell proliferation reported in many organs by other investigators, and the observed results might have been caused by physiological or endocrine mechanisms affecting the epithelium in these calorie-restricted animals.

Macrophage skewing by Phd2 haplodeficiency prevents ischaemia by inducing arteriogenesis.

PHD2 serves as an oxygen sensor that rescues blood supply by regulating vessel formation and shape in case of oxygen shortage. However, it is unknown whether PHD2 can influence arteriogenesis. Here we studied the role of PHD2 in collateral artery growth by using hindlimb ischaemia as a model, a process that compensates for the lack of blood flow in case of major arterial occlusion. We show that Phd2 (also known as Egln1) haplodeficient (Phd2(+/-)) mice displayed preformed collateral arteries that preserved limb perfusion and prevented tissue necrosis in ischaemia. Improved arteriogenesis in Phd2(+/-) mice was due to an expansion of tissue-resident, M2-like macrophages and their increased release of arteriogenic factors, leading to enhanced smooth muscle cell (SMC) recruitment and growth. Both chronic and acute deletion of one Phd2 allele in macrophages was sufficient to skew their polarization towards a pro-arteriogenic phenotype. Mechanistically, collateral vessel preconditioning relied on the activation of canonical NF-κB pathway in Phd2(+/-) macrophages. These results unravel how PHD2 regulates arteriogenesis and artery homeostasis by controlling a specific differentiation state in macrophages and suggest new treatment options for ischaemic disorders.

Profilometric and morphometric response of murine skin to cosmeceutical agents.

OBJECTIVE: To investigate whether topical antiaging compounds can reduce wrinkle depth as noted at replica profilometry with comparable changes in histologic findings in hairless mice. METHODS: Commercial retinoic acid cream, a peptide lotion, and a soy cream were applied to the dorsal skin for 4 weeks. Silicone-negative replicas of treated and untreated skin surface were photographed and evaluated for traditional features of surface roughness. Skin samples were processed using histomorphometry and immunohistochemistry of proliferating cell nuclear antigen. Quantitative light microscopic data were acquired for estimating replication of epidermal keratinocytes, epidermal thickness, and depth of dermal collagen bundles. RESULTS: Data were analyzed by comparing means with 1-way analysis of variance, and significant changes in all measurements were noted. Augmented keratinocyte proliferation and thickening of viable epidermis were observed with all 3 compounds, although a greater effect was found in the retinoic acid and peptide treatment groups. A similar trend was noted with respect to widening of the collagen layer. Epidermal surface roughness manifested maximum smoothing after treatment with the peptide compound. CONCLUSION: The pronounced effects noted with all 3 compounds indicate that topical agents other than retinoic acid may have comparative stimulating effects on the skin in nonirradiated mice.

Comparison of epidermal morphologic response to commercial antiwrinkle agents in the hairless mouse.

BACKGROUND: A large number of commercial antiwrinkle and antiaging compounds are available to consumers for rejuvenation of facial skin ravaged by age or solar radiation. Experimental data on the histological effects of these commercial products in laboratory models are sparse. OBJECTIVES: To compare the efficacy of topical application of five commercially available antiaging compounds (retinoic acid, glycolic acid, vitamin C, estrogen, and soy) on the dorsal skin. METHODS AND MATERIALS: The effects were examined using light microscopic analysis of the epidermis in the normal nonirradiated hairless mouse. The agents were applied daily to dorsal tattooed areas for 2 weeks before histological assessment; neighboring untreated surface areas were used as control. Morphometric measurements of total epidermal width, nuclear volume of keratinocytes in three layers, and index of proliferating cell nuclear antigen according to immunohistochemistry were obtained and statistically analyzed. RESULTS: Significant histomorphometric effects were noticed with all five agents, but more pronounced changes were obtained with glycolic acid, estrogen, and retinoic acid product. CONCLUSIONS: These baseline data will be useful for future studies on the effect of ultraviolet radiation to cause photoaging and reparative effects of similar agents in this animal. The information contained in the report may provide guidelines to consumers and clinicians.

Modulation of cutaneous aging with calorie restriction in Fischer 344 rats: a histological study.

OBJECTIVE: To examine whether histological changes in skin owing to intrinsic aging in a laboratory rodent model are modulated by caloric restriction (CR). METHODS: The abdominal skin from colony-raised ad libitum-fed Fischer 344 rats and age-matched rats subjected to CR was studied in the light microscope using histological morphometric methods. Animals 4, 12, and 24 months or older were used in this study. We studied the skin to obtain (1) quantitative data on the depth of the epidermis, dermis, and fat layer, the epidermal cellular density, the percentage fraction of dermal collagen, elastic fibers, pilosebaceous units, and capillaries, and the fibroblast density; and (2) qualitative assessment of histological staining for dermal glycosaminoglycans. We analyzed data by means of general linear model 2-way analysis of variance to obtain significance for the effects of age, diet, and age-diet interaction. RESULTS: The ad libitum-fed rats showed age-related increase in the depth of the epidermis, dermis, and fat layer. Calorie restriction prevented these changes, but epidermal nuclear density appeared to be stimulated. A trend toward increased values for collagen and elastic fibers, fibroblasts, and capillaries in skin samples from CR rats was observed. Pilosebaceous units were not modified. Moderately reduced staining for the dermal glycosaminoglycans in the skin of CR rats was noticed. CONCLUSIONS: Histomorphological changes resulting from intrinsic aging affected some of the studied variables in the rat skin, and these changes were delayed or prevented by CR. Some stimulatory effects, such as increased densities of fibroblasts and capillary profiles and higher values of connective tissue fibers resulting from CR, were also observed. Cutaneous morphological changes due to natural aging in this rat model seem to be modified by physiological or metabolic alterations imposed by CR.

Use of adenoviruses encoding CD40L or IL-2 against B cell lymphoma.

Some B cell lymphomas lack important costimulatory properties that could prevent them from being used as cell based vaccines. Infection of A20 B lymphoma cells with a replication-defective adenovirus encoding murine (m) CD40L, but not mIL-2, produces an antigen presentation phenotype with upregulation of MHC Class I/II, induction of B7-1/2 molecules and production of MIL-12 and MIP-1alpha. Subcutaneous vaccination with irradiated Ad-mCD40L-infected- or Ad-mIL-2-infected-A20 cells generated A20-specific CD8+ T cell responses and cross reactive A20 Ig antibodies. Only vaccination with Ad-mCD40L-infected A20 cells produced a significant delay in tumor growth and long-term survival (p = 0.0039). Stronger protective immunity to A20 challenge was generated by intravenous priming with A20 cells infected with Ad-mCD40L, Ad-mIL-2 or their combination followed by a boost immunization with A20 cells activated with syngeneic fibroblasts expressing CD40L. Compared to Ad-LacZ-infected A20 priming, the combination priming was most effective followed by Ad-mCD40L and Ad-mIL-2 (p = 0.0027, p = 0.0027, p = 0.0163 respectively). Significant A20-specific CD8+ T cell-mediated cytotoxicity was only demonstrated in splenocytes from these groups of vaccinated animals. By contrast, ELISPOT assay of splenocytes from all A20 prime/boosted vaccinated groups demonstrated increases in gamma-interferon release by T cells elicited by in vitro stimulation either with A20 cells or another syngeneic 2PK-3 lymphoma, indicating the presence of cross reactive immunity. Similarly anti-A20 immunoglobulin antibodies generated after vaccination were not necessarily A20 idiotype-specific. Direct therapy of pre-established tumors was achieved with the combination of Ad-mCD40L and Ad-mIL-2 given at Days 4 and 8 at the tumor site with a significant long-term survival of 85% of tumor-bearing mice (p = 0.0001). Our study strongly supports the use of Ad-CD40L and Ad-IL-2 combination therapy for the treatment of patients with B cell lymphoma.

Fibrin tissue adhesive and autologous concha cartilage for reconstruction of the posterior-superior canal wall of the chinchilla middle ear.

HYPOTHESIS: We conducted this study to prove that fibrin tissue adhesive (FTA) is safe, efficacious, biocompatible, and readily biodegradable with no deleterious side effects for fixation of a cartilage graft to bone along the chinchilla canal wall. METHODS: A posterior-superior canal defect was created in 12 chinchillas. The canal walls of six chinchillas were closed with autologous concha cartilage alone, whereas the canal wall of the remaining six animals were closed with cartilage in conjunction with fibrin tissue adhesive. RESULTS: Animals were killed 8 weeks postoperatively. Three of six cartilage grafts were displaced in the graft alone group, whereas all six grafts in the cartilage with FTA group healed without displacement. CONCLUSION: Fibrin tissue adhesive was found to be effective, biocompatible, biodegradable, and without any deleterious side effects for reconstruction of the superior-posterior canal wall of chinchillas.

Adoptive transfer of anti-idiotypic T cells cure mice of disseminated B cell lymphoma.

There is extensive interest in idiotypic vaccination as a treatment of lymphoma. An alternative approach is the adoptive transfer of in vitro generated T cells. This strategy has been used to treat posttransplantation EBV-related diseases. The ability to generate in vitro T cells to peptides derived from immunoglobulin idiotypes raises the possibility of directly using such cells as a treatment of lymphoma. Investigating the adoptive transfer of specific T cells to idiotype derived peptides in a murine lymphoma model is therefore an important part of the clinical translation of this alternative approach. We have generated an idiotype-specific T cell line, able to recognise a defined, naturally processed idiotype-derived epitope. This line has been used to successfully treat mice with disseminated lymphoma supporting the clinical use of idiotype specific T cells.

Histomorphologic changes in aging skin: observations in the CBA mouse model.

BACKGROUND: Aging of human skin is a phenomenon resulting from a combination of chronological aging and environmental stressors such as sunlight. OBJECTIVES: To study the effects of intrinsic aging on the skin in laboratory-raised CBA mice in 3 age groups, and to assess histological alterations as a function of age in this model. METHODS: Skin samples from CBA mice in 3 age groups (young, young adult, and old) were obtained from the dorsal and ventral areas, pinna, and hind foot to study the following variables using light microscopic manual morphometric methods: the depth of the epidermis and number of epidermal cells, depth of the dermis, and percentage area of dermal collagen, elastic fibers, pilosebaceous units, blood vessels, and tissue space. The obtained values were analyzed using 1-way analysis of variance to detect any significant effects of age. RESULTS: There was a notable attrition of the epidermal thickness and number of cells that could be correlated with age. A reduced number of pilosebaceous units was noted in skin samples from the dorsal region and the footpad. No conspicuous change was noted in the depth of the dermis or percentage area of collagen in aging animals. A proliferation of stainable elastic fibers was demonstrated in the dorsal skin and footpad of older mice. CONCLUSIONS: CBA mice show unique age-related histological modifications of the skin that are different from other rodent species. These baseline data will be helpful in further studies of regenerative effects of pharmaceutical agents on the histological structure of skin and in photoaging studies.

Histological evaluations following 1,064-nm Nd:YAG laser resurfacing.

BACKGROUND AND OBJECTIVES: The long pulse 1,064-nm Nd:YAG laser is used clinically to decrease rhytid formation. The dermal level at which this change occurs has not been established. This study attempts to answer these questions using a porcine skin model. STUDY DESIGN/MATERIALS AND METHODS: Non-randomized prospective experimental trial involving the domestic piglet treated serially with the long pulse 1,064-nm Nd:YAG laser. RESULTS: Collagen formation occurred at the level of the reticular dermis. After one laser treatment, a significant level of collagen formation was induced in the reticular dermis compared to controls. The greatest gain was observed after four laser treatments. Energy levels of 20, 30, 40, and 50 J/cm2 were evaluated. Although not statistically significant, 30 J/cm2 had the greatest effect on collagen formation. However, at 50 J/cm2, marked ablative changes to the epidermis were observed. CONCLUSIONS: The long pulse 1,064-nm Nd:YAG laser induces collagen formation in the reticular dermis in porcine skin.

Immunization with a recombinant adenovirus encoding a lymphoma idiotype: induction of tumor-protective immunity and identification of an idiotype-specific T cell epitope.

The Ig Id of a B cell lymphoma is a tumor-specific Ag, although as a self-Ag it is likely to be a weak immunogen. Provision of a foreign gene may enhance the immunogenicity of the idiotype. Viral vectors allow highly efficient transfer of genetic material and are themselves innately immunogenic. We have investigated the ability of recombinant adenoviral vectors, encoding the idiotypic gene with or without fusion to the human Fc region, to produce anti-idiotypic Ab- and T cell-mediated responses in a syngeneic BALB/c A20 murine lymphoma model. The idiotypic V(H) and V(L) sequences were assembled as a single chain variable fragment (scFv) and adenoviral vectors encoding the A20 scFv (Ad.A20) and A20 scFv linked to the Fc fragment of human IgG1 (Ad.A20hFc) were constructed. A single immunization of BALB/c mice with Ad.A20hFc but not Ad.A20 induced a specific anti-idiotypic Ab response. T cell lines generated from mice vaccinated with either vector displayed specific cytotoxicity, proliferation, and IFN-gamma release against a syngeneic dendritic cell line transduced using a retroviral vector to express the A20 scFv idiotype (XS52.A1.A20). Importantly, both T cell lines lysed the A20 lymphoma cells. An immunodominant H-2K(d)-restricted CD8(+) T cell peptide, DYWGQGTEL (A20[106-114]), was identified as a naturally occurring A20 scFv epitope. A single immunization with Ad.A20hFc but not Ad.A20 provided protection in >40% of animals challenged with a lethal dose of the A20 tumor line and was more effective, in this model, than a previously optimized plasmid vaccine.