Time-dependent appearance of myofibroblasts in granulation tissue of human skin wounds

Human skin wounds (66) inflicted between 20 h and 7 months prior to biopsy were studied. In order to identify the type of cellular differentiation of the fibroblastic cells in the granulation tissue, alpha-smooth muscle actin and desmin were immunohistochemically localized. The value of any presumed time-dependent appearance and/or disappearance of positively stained cells was tested for the estimation of wound age. In skin specimens with a wound age less than 5 days (n =15) no typical granulation tissue had developed and no alpha-actin-positive myofibroblasts could be detected. The first appearance of positively reacting myofibroblasts was noted in a 5-day-old wound. In 57% of the lesions with a wound age between 5 and 31 days (25 out of 44 cases) typical granulation tissue formation was present and myofibroblasts with positive reaction for alpha-smooth muscle actin could be identified. Numerous positively reacting cells could generally be found in wounds aged between 16 and 31 days, but also in wounds less than 16 days old. In 29% of the cases with a wound age of more than 31 days (2 out of 7 cases) alpha-sma-positive myofibroblasts also occured. Fibroblastic cells positive for desmin could not be seen at all in our series. Our results demonstrate the appearance of alpha-sma-positive myofibroblasts with the initial formation of typical granulation tissue in human skin lesions as early as approximately 5 days after wounding. In contrast to recent experimental results these cells remained detectable in wounds aged more than 2 months in some cases. The immunohistochemical detection of actin-positive cells, therefore, demonstrates whether an unknown skin wound is aged approximately 5 days or more. Even though a time-dependent decrease of myofibroblasts in human granulation tissue after 31 days in human wounds seems probable, the extended presence (up to about 2 months) of these cells allows no further exact age determination of older wounds

The time-dependent localization of Ki 67 antigen-positive cells in human skin wounds

A total of 77 human skin wounds with a post-infliction interval between 3 h and 7 months were investigated and the proliferation marker antigen Ki 67 was visualized in paraffin sections using a specific monoclonal antibody (MIB). The re-built epidermal layer covering the former lesional area showed only a few basal cells positively staining for Ki 67 antigen. No enhanced reactivity was found when compared to uninjured skin. In basal cells of the epidermis adjacent to the wound area, however, varying numbers of positive cells occurred, but no information useful for a reliable time estimation of skin wounds could be obtained due to the considerable variability in the number of Ki 67 positive epidermal basal cells found in non-damaged skin. Fibroblastic cells in the wound area revealed an increased number of Ki 67-positive sites which could first be detected in a 1.5-day-old skin lesion. Positive results could be obtained in every specimen investigated after a post-infliction interval of 6 days up to 1.5 months. Only the scar tissue of the oldest wound examined (wound age 7 months) revealed no increase in the number of positively staining fibroblasts. Therefore, positive results indicate a wound age of at least approximately 1.5 days and the lack of an increased number of positive fibroblastic cells in a sufficient number of specimens indicates at a wound age of less than 6 days, but cannot totally exclude longer post-infliction intervals

Immunohistochemical localization of collagen types I and VI in human skin wounds

A total of 74 human skin wounds were investigated and collagen types I and VI were localized in the wound area by immunohistochemistry. Collagen type I appeared in the form of ramifying string-like structures after approximately 5–6 days, but positive reactions in the form of a spot-like staining around isolated fibroblasts also occurred in a skin wound aged 4 days. Collagen VI was detectable after a post-infliction interval of at least 3 days showing a strongly positive reacting network associated with fibroblasts in the wound area. Both collagens appeared almost constantly after a wound age of 6–7 clays and could also be found in wounds aged a few months. Therefore, although a positive reaction for collagen type I in the form of string-like and ramifying structures around wound fibroblasts indicates a wound age of at least 5–6 days, a spot-like positive staining for collagen type I cannot exclude a wound age of at least 4 days. A positive staining for collagen type VI represents a post-infliction time of 3 days or more. The almost constant appearance of these collagen types suggests that negative results in a sufficient number of specimens indicate a wound age of less than 6–7 days, but cannot completely exclude longer post-infliction intervals. Since collagen type I and VI are also found in the granulation/scar tissue of lesions with advanced wound age, the immunohistochemical analysis of these proteins provides no further information for an age determination of older skin wounds

The time-dependent expression of keratins 5 and 13 during the reepithelialization of human skin wounds

The time-dependent reepithelialization of 55 human surgical skin wounds with a wound age between 8h and more than 2 months was investigated by the immunohistochemical localization of cytokeratins 5 and 13. A complete, rebuilt epidermal layer over the wound area was first detectable in a 5-day-old wound, while all wounds of more than 18 days duration contained a completely reepithelialized wound area. Between 5 and 18 days the basal layer of keratinocytes showed — in contrast to normal skin — only some cells positive for cytokeratin 5. In some, but not all lesions with a wound age of 13 days or more, a basal cell layer completely staining for cytokeratin 5 was demonstrable. This staining pattern was found in all skin wounds with a wound age of more than 23 days. The immunohistochemical detection of cytokeratin 13 which can be observed regularly in non-cornifying squamous epithelia provides no information for the time-estimation of human skin wounds, since no significant temporary expression of this polypeptide seems to occur during the healing of human skin wounds

The time-dependent rearrangement of the epithelial basement membrane in human skin wounds

In 62 human skin wounds (surgical wounds, stab wounds and lacerations after surgical treatment) we analyzed the immunohistochemical localization of collagen IV in the epithelial basement membrane. In 27 of these wounds the distribution of collagen VII, which represents a specific component of the basement membrane of stratified epithelia, was also analyzed. We were able to demonstrate a virtually identical co-distribution of both collagen IV and VII in the wound area with no significant time-dependent differences in the appearance of both collagen types. Fragments of the epithelial basement membrane could be detected in the wound area from as early as 4 days after wounding and after 8 days a complete restitution of the epithelial basement membrane was observed. In all cases with a wound age of more than 21 days the basement membrane was completely reformed over the former lesional area. The period between 8 and 21 days after wounding was characterized by a wide variability ranging from complete restitution to deposition of basement membrane fragments or total lack of the epidermal basement membrane

Methods for confidence interval estimation of a ratio parameter with application to location quotients

BACKGROUND: The location quotient (LQ) ratio, a measure designed to quantify and benchmark the degree of relative concentration of an activity in the analysis of area localization, has received considerable attention in the geographic and economics literature. This index can also naturally be applied in the context of population health to quantify and compare health outcomes across spatial domains. However, one commonly observed limitation of LQ is its widespread use as only a point estimate without an accompanying confidence interval. METHODS: In this paper we present statistical methods that can be used to construct confidence intervals for location quotients. The delta and Fieller's methods are generic approaches for a ratio parameter and the generalized linear modelling framework is a useful re-parameterization particularly helpful for generating profile-likelihood based confidence intervals for the location quotient. A simulation experiment is carried out to assess the performance of each of the analytic approaches and a health utilization data set is used for illustration. RESULTS: Both the simulation results as well as the findings from the empirical data show that the different analytical methods produce very similar confidence limits for location quotients. When incidence of outcome is not rare and sample sizes are large, the confidence limits are almost indistinguishable. The confidence limits from the generalized linear model approach might be preferable in small sample situations. CONCLUSION: LQ is a useful measure which allows quantification and comparison of health and other outcomes across defined geographical regions. It is a very simple index to compute and has a straightforward interpretation. Reporting this estimate with appropriate confidence limits using methods presented in this paper will make the measure particularly attractive for policy and decision makers

Improved Statistics for Genome-Wide Interaction Analysis

Recently, Wu and colleagues [1] proposed two novel statistics for genome-wide interaction analysis using case/control or case-only data. In computer simulations, their proposed case/control statistic outperformed competing approaches, including the fast-epistasis option in PLINK and logistic regression analysis under the correct model; however, reasons for its superior performance were not fully explored. Here we investigate the theoretical properties and performance of Wu et al.'s proposed statistics and explain why, in some circumstances, they outperform competing approaches. Unfortunately, we find minor errors in the formulae for their statistics, resulting in tests that have higher than nominal type 1 error. We also find minor errors in PLINK's fast-epistasis and case-only statistics, although theory and simulations suggest that these errors have only negligible effect on type 1 error. We propose adjusted versions of all four statistics that, both theoretically and in computer simulations, maintain correct type 1 error rates under the null hypothesis. We also investigate statistics based on correlation coefficients that maintain similar control of type 1 error. Although designed to test specifically for interaction, we show that some of these previously-proposed statistics can, in fact, be sensitive to main effects at one or both loci, particularly in the presence of linkage disequilibrium. We propose two new “joint effects” statistics that, provided the disease is rare, are sensitive only to genuine interaction effects. In computer simulations we find, in most situations considered, that highest power is achieved by analysis under the correct genetic model. Such an analysis is unachievable in practice, as we do not know this model. However, generally high power over a wide range of scenarios is exhibited by our joint effects and adjusted Wu statistics. We recommend use of these alternative or adjusted statistics and urge caution when using Wu et al.'s originally-proposed statistics, on account of the inflated error rate that can result

Fitness Ranking of Individual Mutants Drives Patterns of Epistatic Interactions in HIV-1

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