Longitudinal analysis of antibody trajectories and humoral responses to a third dose of mRNA vaccines against SARS-CoV-2 in patients with a history of anti-CD20 therapy (RituxiVac 2.0)

BackgroundMorbidity and mortality of COVID-19 is increased in patients with B-cell depleting therapies, the majority of which also show compromised vaccination-induced immune responses. Herein, we report on the trajectories of anti-SARS-CoV-2 antibodies in patients from the original RituxiVac study compared to healthy volunteers and investigate the immunogenicity of a third vaccination in previously non-responding patients. MethodsA follow-up evaluation was performed in volunteers and patients from the RituxiVac Study (NCT04877496), which investigated the humoral and cell-mediated immune response after SARS-CoV-2 mRNA vaccination in patients with a history with anti-CD20 depleting therapies (rituximab or ocrelizumab). The current population included 33 patients and 26 healthy volunteers with initial humoral vaccine response and 32 non-responding patients. Primary outcome was anti-SARS-CoV-2 antibody trajectories in vaccine responders 4.3 months (median; interquartile range [IQR]: 3.6 - 4.8 months) after first evaluation and humoral responses after a third vaccine dose in previous non-responders. Antibody decay rates were compared using analysis of covariance in linear regression. ResultsIn patients with detectable anti-spike IgG antibodies after two-dose vaccination, circulating anti-spike IgG persisted in 88% (29/33) of patients 5.6 months after the second vaccination (median; IQR: 5.1-6.7) compared to 92% (24/26) of healthy volunteers 6.8 months after the second dose (IQR: 6.0-7.1) (p=0.7). Antibody decay rates were comparable between patients and controls with -0.54 signal/cut-off (s/c) units per month (IQR -0.72 to -0.45) and -0.60 s/c units per month (IQR: -0.88 to -0.44), p=0.70. Two-dose responders with loss of circulating antibodies at follow-up (n=4/33, 12%) had lower initial antibody concentrations (p<0.01). Biomarkers for immunocompetence, including CD3, CD4 or CD19 cell count at baseline did not predict anti-spike IgG persistence. In two-dose non-responders, a third dose of mRNA vaccine against SARS-CoV-2 elicited humoral response with detectable anti-spike IgG antibodies in 19% (6/32) participants. No clinical parameters nor biomarkers of immunocompetence predicted humoral response after a third vaccine dose. ConclusionThe present study reveals comparable antibody reduction rates between patients with CD20-depleting treatment history and healthy volunteers, but inefficient humoral responses to a third dose of SARS-CoV-2 mRNA vaccines in the majority of two-dose non-responders. There is a need for individually tailored vaccination strategies in immunocompromised patients that could be stratified by B cell counts and initial level of antibody titers. (Funded by Bern University Hospital, ClinicalTrials.gov number, NCT04877496)

Endemic Seasonal Coronavirus Neutralisation and COVID-19 severity

The virus SARS-CoV-2, responsible for the global COVID-19 pandemic, spread rapidly around the world causing high morbidity and mortality because humans have no pre-existing immunity. However, there are four known, endemic seasonal coronaviruses in humans (HCoVs) and whether antibodies for these HCoVs play a role in severity of COVID-19 disease has generated a lot of interest. Of these seasonal viruses NL63 is of particular interest as it uses the same cell entry receptor as SARS-CoV-2.We use functional, neutralising assays to investigate cross reactive antibodies and their relationship with COVID-19 severity. We analysed neutralisation of SARS-CoV-2, NL63, HKU1, and 229E in 38 COVID-19 patients and 62 healthcare workers, and a further 182 samples to specifically study the relationship between SARS-CoV-2 and NL63.We found that although HCoV neutralisation was very common there was little evidence that these antibodies neutralised SARS-CoV-2. Despite no evidence in cross neutralisation, levels of NL63 neutralisating antibodies become elevated after exposure to SARS-CoV-2 through infection or following vaccination.

Pseudotyped Bat Coronavirus RaTG13 is efficiently neutralised by convalescent sera from SARS-CoV-2 infected Patients

RaTG13 is a close relative of SARS-CoV-2, the virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, sharing 96% sequence similarity at the genome-wide level. The spike receptor binding domain (RBD) of RaTG13 contains a large number of amino acid substitutions when compared to SARS-CoV-2, likely impacting affinity for the ACE2 receptor. Antigenic differences between the viruses are less well understood, especially whether RaTG13 spike can be efficiently neutralised by antibodies generated from infection with, or vaccination against, SARS-CoV-2. Using RaTG13 and SARS-CoV-2 pseudotypes we compared neutralisation using convalescent sera from previously infected patients as well as vaccinated healthcare workers. Surprisingly, our results revealed that RaTG13 was more efficiently neutralised than SARS-CoV-2. In addition, neutralisation assays using spike chimeras and mutants harbouring single amino acid substitutions within the RBD demonstrated that both spike proteins can tolerate multiple changes without dramatically reducing how efficiently they are neutralised. Moreover, introducing the 484K mutation into RaTG13 resulted in increased neutralisation, in contrast to the same mutation in SARS-CoV-2 (E484K). This is despite E484K having a well-documented role in immune evasion in variants of concern (VOC) such as B.1.351 (Beta). These results indicate that the immune-escape mutations found in SARS-CoV-2 VOCs might be driven by strong antibody pressures, and that the future spill-over of RaTG13 and/or related sarbecoviruses could be mitigated using current SARS-CoV-2-based vaccination strategies.

Humoral and cellular responses to mRNA vaccines against SARS-CoV2 in patients with a history of CD20-B-cell depleting therapy

BackgroundB-cell depleting therapies increase COVID19 morbidity and mortality. For this specific population, evidence-based vaccination strategies are lacking. Here, we investigated humoral and cell mediated immune responses to SARS-CoV2 mRNA-based vaccines in patients receiving CD20-B-cell depleting agents for autoimmune disease, malignancy, or transplantation. MethodsPatients at the Bern University Hospital with a treatment history of anti-CD20 depleting agents (rituximab or ocrelizumab) were enrolled for analysis of humoral and cell-mediated immune responses (by interferon-{gamma} release assay) after completing vaccination against SARS-CoV2. Primary outcome was the the anti-spike antibody response in anti-CD20-treated patients (n=96) in comparison to immunocompetent controls (n=29). ResultsAnti-spike IgG antibodies were detected in 49% of patients 1.79 months after the second vaccine dose (interquartile range, IQR: 1.16-2.48) compared to 100% of controls (p<0.001). SARS-CoV2 specific interferon-{gamma} release was detected in 20% of patients and 75% of healthy controls (p<0.001). Only 11% of patients, but 75%of healthy controls showed positive reactions in both assays, respectively (p<0.001). Time since last anti-CD20 therapy (7.6 months), peripheral CD19+ (>27/{micro}l), and CD4+ lymphocyte count (>653/{micro}l) predicted humoral vaccine response (area under the curve [AUC]: 67% [CI 56-78], 67% [CI 55-80] and 66% [CI 54-79], (positive predictive value [PPV]: 0.78, 0.7 and 0.71). ConclusionThis study provides evidence for blunted humoral and cell-mediated immune responses elicited by SARS-CoV2 mRNA vaccines in patients with CD20-depleting treatment history. Lymphocyte subpopulation counts are associated with vaccine response in this highly vulnerable population. (Funded by Bern University Hospital, ClinicalTrials.gov number, NCT04877496)

Breadth of neutralising antibody responses to SARS-CoV-2 variants of concern is augmented by vaccination following prior infection: studies in UK healthcare workers and immunodeficient patients

Approximately 75% of the UK population has received only one dose of a 2-dose COVID-19 vaccine regime in the face of circulating SARS-CoV-2 Variants of Concern (VOCs). We aimed to determine the levels of vaccine-induced neutralising antibodies to SARS-CoV-2 variants B.1.1.7, B.1.351 and P.1. To do so, we undertook a single-centre cross-sectional study of health care workers (HCWs) and outpatients with immunodeficiencies (IDP) based at the same critical care tertiary NHS Trust, following a single dose of either BNT162b2 or AZD1222 vaccines. Data revealed low neutralising antibodies (nAbs) in IDPs, with only 5% and 3% showing detectable neutralisation of B.1.1.7 and B.1.351, respectively. In contrast, healthy HCWs without a prior SARS-CoV-2 infection demonstrated a wide range of nAb titres post-vaccination with responses significantly lower than HCWs with prior SARS-CoV-2 infection. Neutralisation of VOCs with the E484K mutation (B.1.351 and P.1) were consistently lower in HCWs in the absence of evidence of prior SARS-CoV-2 infection (p<0.001). Notably, in vaccinated HCWs with prior SARS-CoV-2 infection, there was a significant increase of neutralising titres post-vaccination to all variants, compared to their pre-vaccination neutralisation titres. This underscores the importance of vaccination to boost neutralising antibody breadth to VOCs, and also provides support for the hypothesis that repeated immunisations will boost protective immunity in individuals without prior SARS-CoV-2 exposure.

Standardised, quantitative neutralisation responses to SARS-CoV-2 Variants of Concern by convalescent anti-sera from first wave infections of UK Health Care Workers and Patients

BackgroundThe rise of SARS-CoV-2 variants has made the pursuit to define correlates of protection more troublesome, despite the availability of the World Health Organisation (WHO) International Standard for anti-SARS-CoV-2 Immunoglobulin sera, a key reagent used to standardise laboratory findings into an international unitage. MethodsUsing pseudotyped virus, we examine the capacity of convalescent sera, from a well-defined cohort of healthcare workers (HCW) and Patients infected during the first wave from a national critical care centre in the UK to neutralise B.1.1.298, variants of interest (VOI) B.1.617.1 (Kappa), and four VOCs, B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta), including the B.1.617.2 K417N, informally known as Delta Plus. We utilised the WHO International Standard for anti-SARS-CoV-2 Immunoglobulin to report neutralisation antibody levels in International Units per mL. FindingsOur data demonstrate a significant reduction in the ability of first wave convalescent sera to neutralise the VOCs. Patients and HCWs with more severe COVID-19 were found to have higher antibody titres and to neutralise the VOCs more effectively than individuals with milder symptoms. Using an estimated threshold for 50% protection, 54 IU/mL, we found most asymptomatic and mild cases did not produce titres above this threshold. InterpretationExpressing our data in IU/ml, we provide a benchmark pre-vaccine standardised dataset that compares disease severity with neutralising antibody titres. Our data may now be compared across multiple laboratories. The continued use and aggregation of standardised data will eventually assist in defining correlates of protection. FundingUKRI and NIHR; grant number G107217 Research in contextO_ST_ABSEvidence before this studyC_ST_ABSDuring the first wave outbreak, much focus was placed on the role of neutralising antibodies and titres generated upon infection to ancestral SARS-CoV-2. Due to the large amounts of different assays used to elucidate the antibody-mediated immunity and laboratory to laboratory, large amounts of invaluable data could not be directly compared in order to define a correlate of protection, due to variability in the results. The WHO International Standard for anti-SARS-CoV-2 Immunoglobulin sera was made in order to standardise future data so that comparisons may take place. Added value of this studyOur study compares the neutralisation capacity of sera from patients and healthcare workers (HCWs) from the ancestral strain of SARS-CoV-2 against new variants, including the current variants of concern in circulation. We also provide data in International Units per mL, a standardised unitage, for infected individuals that have a clinical severity score, allowing us to assess levels of neutralising antibodies across different severities of COVID-19 disease. By providing a method to calibrate most of the variants of concern so that the WHO International Standard for anti-SARS-CoV-2 Immunoglobulin reagent could be used to standardise our results, therefore making them comparable to other laboratories who also standardised their data in an identical manner. Implications of all the available evidenceContinual use and accumulation of standardised data would eventually lead to defining the correlates of protection against SARS-CoV-2. This could help to inform medical staff to identify which individuals would be a greater risk of a potential reinfection to SARS-CoV-2.

Towards Internationally standardised humoral Immune Correlates of Protection from SARS CoV 2 infection and COVID-19 disease

Precision monitoring of antibody responses during the COVID-19 pandemic is increasingly important during large scale vaccine rollout and rise in prevalence of Severe Acute Respiratory Syndrome-related Coronavirus-2 (SARS-CoV-2) variants of concern (VOC). Equally important is defining Correlates of Protection (CoP) for SARS-CoV-2 infection and COVID-19 disease. Data from epidemiological studies and vaccine trials identified virus neutralising antibodies (Nab) and SARS-CoV-2 antigen-specific (notably RBD, and S) binding antibodies as candidate CoP. In this study, we used the World Health Organisation (WHO) international standard to benchmark neutralising antibody responses and a large panel of binding antibody assays to compare convalescent sera obtained from: a) COVID-19 patients; b) SARS-CoV-2 seropositive healthcare workers (HCW) and c) seronegative HCW. The ultimate aim of this study, was to identify biomarkers of humoral immunity that could be used as candidate CoP in internationally accepted unitage. Whenever suitable, the antibody levels of the samples studied were expressed in International Units (INU) for virus neutralisation assays or International Binding Antibody Units (BAU) for ELISA tests. In this work we used commercial and non-commercial antibody binding assays; a lateral flow test for detection of SARS-CoV-2-specific IgG / IgM; a high throughput multiplexed particle flow cytometry assay for SARS-CoV-2 Spike (S), Nucleocapsid (N) and Receptor Binding Domain (RBD) proteins); a multiplex antigen semi-automated immuno-blotting assay measuring IgM, IgA and IgG; a pseudotyped microneutralisation test (pMN) and electroporation-dependent neutralisation assay (EDNA). Our results indicate that overall, severe COVID-19 patients showed statistically significantly higher levels of SARS-CoV-2-specific neutralising antibodies (average 1029 IU/ml) than those observed in seropositive HCW with mild or asymptomatic infections (379 IU/ml) and that clinical severity scoring, based on WHO guidelines was tightly correlated with neutralisation and RBD / S binding assays. In addition, there was a positive correlation between severity, N-antibody assays and intracellular virus neutralisation.

Knowledge barriers in the symptomatic-COVID-19 testing programme in the UK: an observational study

BackgroundSymptomatic testing programmes are crucial to the COVID-19 pandemic response. We sought to examine United Kingdom (UK) testing rates amongst individuals with test-qualifying symptoms, and factors associated with not testing. MethodsWe analysed a cohort of untested symptomatic app users (N=1,237), nested in the Zoe COVID Symptom Study (Zoe, N= 4,394,948); and symptomatic survey respondents who wanted, but did not have a test (N=1,956), drawn from the University of Maryland-Facebook Covid-19 Symptom Survey (UMD-Facebook, N=775,746). FindingsThe proportion tested among individuals with incident test-qualifying symptoms rose from [~]20% to [~]75% from April to December 2020 in Zoe. Testing was lower with one vs more symptoms (73.0% vs 85.0%), or short vs long symptom duration (72.6% vs 87.8%). 40.4% of survey respondents did not identify all three test-qualifying symptoms. Symptom identification decreased for every decade older (OR=0.908 [95% CI 0.883-0.933]). Amongst symptomatic UMD-Facebook respondents who wanted but did not have a test, not knowing where to go was the most cited factor (32.4%); this increased for each decade older (OR=1.207 [1.129-1.292]) and for every 4-years fewer in education (OR=0.685 [0.599-0.783]). InterpretationDespite current UK messaging on COVID-19 testing, there is a knowledge gap about when and where to test, and this may be contributing to the [~]25% testing gap. Risk factors, including older age and less education, highlight potential opportunities to tailor public health messages. FundingZoe Global Limited, Department of Health, Wellcome Trust, EPSRC, NIHR, MRC, Alzheimers Society, Facebook Sponsored Research Agreement. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSTo assess current evidence on test uptake in symptomatic testing programmes, and the reasons for not testing, we searched PubMed from database inception for research using the keywords (COVID-19) AND (testing) AND ((access) OR (uptake)). We did not find any work reporting on levels of test uptake amongst symptomatic individuals. We found three papers investigating geographic barriers to testing. We found one US based survey reporting on knowledge barriers to testing, and one UK based survey reporting on barriers in the period March - August 2020. Neither of these studies were able to combine testing behaviour with prospectively collected symptom reports from the users surveyed. Added value of this studyThrough prospective collection of symptom and test reports, we were able to estimate testing uptake amongst individuals with test-qualifying symptoms in the UK. Our results indicate that whilst testing has improved since the start of the pandemic, there remains a considerable testing gap. Investigating this gap we find that individuals with just one test-qualifying symptom or short symptom duration are less likely to get tested. We also find knowledge barriers to testing: a substantial proportion of individuals do not know which symptoms qualify them for a COVID-19 test, and do not know where to seek testing. We find a larger knowledge gap in individuals with older age and fewer years of education. Implications of all the available evidenceDespite the UK having a simple set of symptom-based testing criteria, with tests made freely available through nationalised healthcare, a quarter of individuals with qualifying symptoms do not get tested. Our findings suggest testing uptake may be limited by individuals not acting on mild or transient symptoms, not recognising the testing criteria, and not knowing where to get tested. Improved messaging may help address this testing gap, with opportunities to target individuals of older age or fewer years of education. Messaging may prove even more valuable in countries with more fragmented testing infrastructure or more nuanced testing criteria, where knowledge barriers are likely to be greater.

SARS-CoV-2 Viral Load is Associated with Increased Disease Severity and Mortality

The relationship between SARS-CoV-2 viral load and risk of disease progression remains largely undefined in coronavirus disease 2019 (COVID-19). We quantified SARS-CoV-2 viral load from participants with a diverse range of COVID-19 severity, including those requiring hospitalization, outpatients with mild disease, and individuals with resolved infection. SARS-CoV-2 plasma RNA was detected in 27% of hospitalized participants and 13% of outpatients diagnosed with COVID-19. Amongst the participants hospitalized with COVID-19, higher prevalence of detectable SARS-CoV-2 plasma viral load was associated with worse respiratory disease severity, lower absolute lymphocyte counts, and increased markers of inflammation, including C-reactive protein and IL-6. SARS-CoV-2 viral loads, especially plasma viremia, were associated with increased risk of mortality. SARS-CoV-2 viral load may aid in the risk stratification of patients with COVID-19 and its role in disease pathogenesis should be further explored.

Attenuated total reflection Fourier-transform infrared (ATR-FTIR) imaging of tissues and live cells.

FTIR spectroscopic imaging is a label-free, non-destructive and chemically specific technique that can be utilised to study a wide range of biomedical applications such as imaging of biopsy tissues, fixed cells and live cells, including cancer cells. In particular, the use of FTIR imaging in attenuated total reflection (ATR) mode has attracted much attention because of the small, but well controlled, depth of penetration and corresponding path length of infrared light into the sample. This has enabled the study of samples containing large amounts of water, as well as achieving an increased spatial resolution provided by the high refractive index of the micro-ATR element. This review is focused on discussing the recent developments in FTIR spectroscopic imaging, particularly in ATR sampling mode, and its applications in the biomedical science field as well as discussing the future opportunities possible as the imaging technology continues to advance.

Cardiotoxicity of Mitoxantrone Treatment in a German Cohort of 639 Multiple Sclerosis Patients

BACKGROUND AND PURPOSE: The aim of this study was to elucidate the role of therapy-related cardiotoxicity in multiple sclerosis (MS) patients treated with mitoxantrone and to identify potential predictors for individual risk assessment. METHODS: Within a multicenter retrospective cohort design, cardiac side effects attributed to mitoxantrone were analyzed in 639 MS patients at 2 MS centers in Germany. Demographic, disease, treatment, and follow-up data were collected from hospital records. Patients regularly received cardiac monitoring during the treatment phase. RESULTS: None of the patients developed symptomatic congestive heart failure. However, the frequency of patients experiencing cardiac dysfunction of milder forms after mitoxantrone therapy was 4.1% (26 patients) among all patients. Analyses of the risk for cardiotoxicity revealed that cumulative dose exposure was the only statistically relevant risk factor associated with cardiac dysfunction. CONCLUSIONS: The number of patients developing subclinical cardiac dysfunction below the maximum recommended cumulative dose is higher than was initially assumed. Interestingly, a subgroup of patients was identified who experienced cardiac dysfunction shortly after initiation of mitoxantrone and who received a low cumulative dose. Therefore, each administration of mitoxantrone should include monitoring of cardiac function to enhance the treatment safety for patients and to allow for early detection of any side effects, especially in potential high-risk subgroups (as determined genetically).

Alleviated affect of exogenous CaCl2 on the growth, antioxidative enzyme activities and cadmium absorption efficiency of Wedelia trilobata hairy roots under cadmium stress / 生物工程学报

In order to study the physiological mechanism of exogenous calcium on the toxicity of heavy metal cadmium (Cd) to Wedelia trilobata hairy roots, the effects of Cd alone, and in combination with different concentrations of Ca on growth, contents of soluble protein and malondialdehyde (MDA), activities of superoxide dismutase (SOD) and peroxidase (POD), Cd2+ absorption in W. trilobata hairy roots were investigated. Cd concentrations lower than 50 micromol/L enhanced the growth of hairy roots, while concentrations higher than 100 micromol/L inhibited growth, making the branched roots short and small, and also turning the root tips brown, even black. In comparison with the control (0 micromol/L Cd), the soluble protein content in hairy roots was found to increase when cultured with 10-50 micromol/L Cd, and decrease when exposed to a cadmium concentration higher than 100 micromol/L Cd. In addition, the activities of POD and SOD activity and MDA content were significantly higher than the control. Compared to the control (hairy roots cultured without 10-30 mmol/L Ca), 100 micromol/L Cd or 300 micromol/L Cd in combination with 10-30 mmol/L Ca resulted in increased growth, causing the main root and secondary roots thicker and also an increase in soluble protein content. On the contrary, MDA content and POD and SOD activities decreased. Quantitative analysis by Atomic Absorption Spectrophotometry showed that W. trilobata hairy roots can absorb and adsorb heavy metal Cd in the ionic form of Cd2+. The maximum content of Cd2+ absorbed by the hairy roots was obtained with a concentration 100 micromol/L Cd2+ while that of Cd2+ adsorbed by hairy roots was achieved with a concentration of 300 micromol/L Cd2+. The exogenous addition of 10-30 mmol/L Ca2+ was found to reduce the absorption, adsorption of Cd2+ and the toxicity of Cd significantly. This reduction in toxicity was caused by the reduction in the absorption of Cd and decreasing the lipid peroxidation through regulating the activities of antioxidant enzymes SOD and POD in the hairy roots.

Raman microspectroscopic and dynamic vapor sorption characterization of hydration in collagen and dermal tissue.

Water is an integral part of collagen's triple helical and higher order structure. Studies of model triple helical peptides have revealed the presence of repetitive intrachain, interchain, and intermolecular water bridges (Bella et al., Structure 1995, 15, 893-906). In addition, an extended cylinder of hydration is thought to be responsible for collagen fiber assembly. Confocal Raman spectroscopy and dynamic vapor sorption (DVS) measurements of human Type I collagen and pigskin dermis were performed to probe relative humidity (RH)-dependent differences in the nature and level of collagen hydration. Raman spectra were also acquired as a function of time for both Type I collagen and pigskin dermis samples upon exchange of a 100% RH H(2) O to deuterium oxide (D(2) O) environment. Alterations in Amide I and III modes were consistent with anticipated changes in hydrogen bonding strength as RH increased and upon H → D exchange. Of note is the identification of a Raman spectral marker (band at 938 cm(-1) ) which appears to be sensitive to alterations in collagen-bound water. Analysis of DVS isotherms provided a quantitative measure of adsorbed and absorbed water vapor consistent with the Raman results. The development of a Raman spectral marker of collagen hydration in intact tissue is relevant to diverse fields of study ranging from the evaluation of therapeutics for wound healing to hydration of aging skin.

A coordinated approach to cutaneous wound healing: vibrational microscopy and molecular biology.

The repair of cutaneous wounds in the adult body involves a complex series of spatially and temporally organized processes to prevent infection and restore homeostasis. Three characteristic phases of wound repair (inflammation, proliferation including re-epithelialization and remodelling) overlap in time and space. We have utilized a human skin wound-healing model to correlate changes in genotype and pheno-type with infrared (IR) and confocal Raman spectroscopic images during the re-epithelialization of excisional wounds. The experimental protocols validated as IR images clearly delineate the keratin-rich migrating epithelial tongue from the collagen-rich wound bed. Multivariate statistical analysis of IR datasets acquired 6 days post-wounding reveal subtle spectral differences that map to distinct spatial distributions, which are correlated with immunofluorescent staining patterns of different keratin types. Images computed within collagen-rich regions expose complementary spatial patterns and identify elastin in the wound bed. The temporal sequence of events is explored through a comparison of gene array analysis with confocal Raman microscopy. Our approach demonstrates the feasibility of acquiring detailed molecular structure information from the various proteins and their subclasses involved in the wound-healing process.

Chemical imaging of the stratum corneum under controlled humidity with the attenuated total reflection Fourier transform infrared spectroscopy method.

Attenuated total reflection Fourier transform infrared spectroscopic imaging was applied to study human stratum corneum (SC) tissue, the outermost layer of the skin. This imaging approach was combined with a controlled environment cell to demonstrate the possibility of obtaining chemical images of SC exposed to a wide range of relative humidities and diffusion of ethanol through the SC tissue with a specially designed liquid cell. The effect of water vapor sorbed into the SC on the distribution of other components in the SC was studied. Principal component analysis was applied in conjunction with univariate analysis to differentiate the distribution of different components in the SC. Swelling of the SC, a heterogeneous distribution of natural moisturizing factor and water, was detected upon the increase of relative humidity. The approach to image the penetration of liquid ethanol into the SC was also demonstrated and showed good potential and implications for studying transdermal drug delivery.