Booster vaccination with Ad26.COV2.S or an Omicron-adapted vaccine in pre-immune hamsters protects against Omicron BA.2.

Since the original outbreak of the SARS-CoV-2 virus, several rapidly spreading SARS-CoV-2 variants of concern (VOC) have emerged. Here, we show that a single dose of Ad26.COV2.S (based on the Wuhan-Hu-1 spike variant) protects against the Gamma and Delta variants in naive hamsters, supporting the observed maintained vaccine efficacy in humans against these VOC. Adapted spike-based booster vaccines targeting Omicron variants have now been authorized in the absence of human efficacy data. We evaluated the immunogenicity and efficacy of Ad26.COV2.S.529 (encoding a stabilized Omicron BA.1 spike) in naive mice and in hamsters with pre-existing immunity to the Wuhan-Hu-1 spike. In naive mice, Ad26.COV2.S.529 elicited higher neutralizing antibody titers against SARS-CoV-2 Omicron BA.1 and BA.2, compared with Ad26.COV2.S. However, neutralizing titers against the SARS-CoV-2 B.1 (D614G) and Delta variants were lower after primary vaccination with Ad26.COV2.S.529 compared with Ad26.COV2.S. In contrast, we found comparable Omicron BA.1 and BA.2 neutralizing titers in hamsters with pre-existing Wuhan-Hu-1 spike immunity after vaccination with Ad26.COV2.S, Ad26.COV2.S.529 or a combination of the two vaccines. Moreover, all three vaccine modalities induced equivalent protection against Omicron BA.2 challenge in these animals. Overall, our data suggest that an Omicron BA.1-based booster in rodents does not improve immunogenicity and efficacy against Omicron BA.2 over an Ad26.COV2.S booster in a setting of pre-existing immunity to SARS-CoV-2.

TRPS1 acts as a context-dependent regulator of mammary epithelial cell growth/differentiation and breast cancer development.

The GATA-type zinc finger transcription factor TRPS1 has been implicated in breast cancer. However, its precise role remains unclear, as both amplifications and inactivating mutations in TRPS1 have been reported. Here, we used in vitro and in vivo loss-of-function approaches to dissect the role of TRPS1 in mammary gland development and invasive lobular breast carcinoma, which is hallmarked by functional loss of E-cadherin. We show that TRPS1 is essential in mammary epithelial cells, since TRPS1-mediated suppression of interferon signaling promotes in vitro proliferation and lactogenic differentiation. Similarly, TRPS1 expression is indispensable for proliferation of mammary organoids and in vivo survival of luminal epithelial cells during mammary gland development. However, the consequences of TRPS1 loss are dependent on E-cadherin status, as combined inactivation of E-cadherin and TRPS1 causes persistent proliferation of mammary organoids and accelerated mammary tumor formation in mice. Together, our results demonstrate that TRPS1 can function as a context-dependent tumor suppressor in breast cancer, while being essential for growth and differentiation of normal mammary epithelial cells.

Exogenous ERα Expression in the Mammary Epithelium Decreases Over Time and Does Not Contribute to p53-Deficient Mammary Tumor Formation in Mice.

Approximately 75% of all breast cancers express the nuclear hormone receptor estrogen receptor α (ERα). However, the majority of mammary tumors from genetically engineered mouse models (GEMMs) are ERα-negative. To model ERα-positive breast cancer in mice, we exogenously introduced expression of mouse and human ERα in an existing GEMM of p53-deficient breast cancer. After initial ERα expression during mammary gland development, expression was reduced or lost in adult glands and p53-deficient mammary tumors. Chromatin immunoprecipitation (ChIP)-sequencing analysis of primary mouse mammary epithelial cells (MMECs) derived from these models, in which expression of the ERα constructs was induced in vitro, confirmed interaction of ERα with the DNA. In human breast and endometrial cancer, and also in healthy breast tissue, DNA binding of ERα is facilitated by the pioneer factor FOXA1. Surprisingly, the ERα binding sites identified in primary MMECs, but also in mouse mammary gland and uterus, showed an high enrichment of ERE motifs, but were devoid of Forkhead motifs. Furthermore, exogenous introduction of FOXA1 and GATA3 in ERα-expressing MMECs was not sufficient to promote ERα-responsiveness of these cells. Together, this suggests that species-specific differences in pioneer factor usage between mouse and human are dictated by the DNA sequence, resulting in ERα-dependencies in mice that are not FOXA1 driven. These species-specific differences in ERα-biology may limit the utility of mice for in vivo modeling of ERα-positive breast cancer.

GATA3 Truncating Mutations Promote Cistromic Re-Programming In Vitro, but Not Mammary Tumor Formation in Mice.

Heterozygous mutations in the transcription factor GATA3 are identified in 10-15% of all breast cancer cases. Most of these are protein-truncating mutations, concentrated within or downstream of the second GATA-type zinc-finger domain. Here, we investigated the functional consequences of expression of two truncated GATA3 mutants, in vitro in breast cancer cell lines and in vivo in the mouse mammary gland. We found that the truncated GATA3 mutants display altered DNA binding activity caused by preferred tethering through FOXA1. In addition, expression of the truncated GATA3 mutants reduces E-cadherin expression and promotes anchorage-independent growth in vitro. However, we could not identify any effects of truncated GATA3 expression on mammary gland development or mammary tumor formation in mice. Together, our results demonstrate that both truncated GATA3 mutants promote cistromic re-programming of GATA3 in vitro, but these mutants are not sufficient to induce tumor formation in mice.

Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity.

Bispecific antibodies have therapeutic potential by expanding the functions of conventional antibodies. Many different formats of bispecific antibodies have meanwhile been developed. Most are genetic modifications of the antibody backbone to facilitate incorporation of two different variable domains into a single molecule. Here, we present a bispecific format where we have fused two full-sized IgG antibodies via their C termini using sortase transpeptidation and click chemistry to create a covalently linked IgG antibody heterodimer. By linking two potent anti-influenza A antibodies together, we have generated a full antibody dimer with bispecific activity that retains the activity and stability of the two fusion partners.

Rapid emergence of a virulent PB2 E627K variant during adaptation of highly pathogenic avian influenza H7N7 virus to mice.

BACKGROUND: Highly pathogenic avian influenza (HPAI) viruses pose a potential human health threat as they can be transmitted directly from infected poultry to humans. During a large outbreak of HPAI H7N7 virus among poultry in The Netherlands in 2003, bird to human transmission was confirmed in 89 cases, of which one had a fatal outcome. METHODS: To identify genetic determinants of virulence in a mammalian host, we passaged an avian H7N7/03 outbreak isolate in mouse lungs and evaluated the phenotype of the mouse-adapted variant in animal models and in vitro. RESULTS: Three passages in mouse lungs were sufficient to select a variant that was highly virulent in mice. The virus had a MLD50 that was >4.3 logs lower than that of its non-lethal parental virus. Sequence analysis revealed a single mutation at position 627 in PB2, where the glutamic acid was changed to a lysine (E627K). The mouse-adapted virus has this mutation in common with the fatal human case isolate. The virus remained highly pathogenic for chickens after its passage in mice. In ferrets, the mouse-adapted virus induced more severe disease, replicated to higher titers in the lower respiratory tract and spread more efficiently to systemic organs compared with the parental virus. In vitro, the PB2 E627K mutation had a promoting effect on virus propagation in mammalian, but not in avian cells. CONCLUSIONS: Our results show that the E627K mutation in PB2 alone can be sufficient to convert an HPAI H7N7 virus of low virulence to a variant causing severe disease in mice and ferrets. The rapid emergence of the PB2 E627K mutant during mouse adaptation and its pathogenicity in ferrets emphasize the potential risk of HPAI H7N7 viruses for human health.

Protective efficacy of Newcastle disease virus expressing soluble trimeric hemagglutinin against highly pathogenic H5N1 influenza in chickens and mice.

BACKGROUND: Highly pathogenic avian influenza virus (HPAIV) causes a highly contagious often fatal disease in poultry, resulting in significant economic losses in the poultry industry. HPAIV H5N1 also poses a major public health threat as it can be transmitted directly from infected poultry to humans. One effective way to combat avian influenza with pandemic potential is through the vaccination of poultry. Several live vaccines based on attenuated Newcastle disease virus (NDV) that express influenza hemagglutinin (HA) have been developed to protect chickens or mammalian species against HPAIV. However, the zoonotic potential of NDV raises safety concerns regarding the use of live NDV recombinants, as the incorporation of a heterologous attachment protein may result in the generation of NDV with altered tropism and/or pathogenicity. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we generated recombinant NDVs expressing either full length, membrane-anchored HA of the H5 subtype (NDV-H5) or a soluble trimeric form thereof (NDV-sH5(3)). A single intramuscular immunization with NDV-sH5(3) or NDV-H5 fully protected chickens against disease after a lethal challenge with H5N1 and reduced levels of virus shedding in tracheal and cloacal swabs. NDV-sH5(3) was less protective than NDV-H5 (50% vs 80% protection) when administered via the respiratory tract. The NDV-sH5(3) was ineffective in mice, regardless of whether administered oculonasally or intramuscularly. In this species, NDV-H5 induced protective immunity against HPAIV H5N1, but only after oculonasal administration, despite the poor H5-specific serum antibody response it elicited. CONCLUSIONS/SIGNIFICANCE: Although NDV expressing membrane anchored H5 in general provided better protection than its counterpart expressing soluble H5, chickens could be fully protected against a lethal challenge with H5N1 by using the latter NDV vector. This study thus provides proof of concept for the use of recombinant vector vaccines expressing a soluble form of a heterologous viral membrane protein. Such vectors may be advantageous as they preclude the incorporation of heterologous membrane proteins into the viral vector particles.

Glycan-dependent immunogenicity of recombinant soluble trimeric hemagglutinin.

Recombinant soluble trimeric influenza A virus (IAV) hemagglutinin (sHA(3)) has proven an effective vaccine antigen against IAV. Here, we investigate to what extent the glycosylation status of the sHA(3) glycoprotein affects its immunogenicity. Different glycosylation forms of subtype H5 trimeric HA protein (sH5(3)) were produced by expression in insect cells and different mammalian cells in the absence and presence of inhibitors of N-glycan-modifying enzymes or by enzymatic removal of the oligosaccharides. The following sH5(3) preparations were evaluated: (i) HA proteins carrying complex glycans produced in HEK293T cells; (ii) HA proteins carrying Man(9)GlcNAc(2) moieties, expressed in HEK293T cells treated with kifunensine; (iii) HA proteins containing Man(5)GlcNAc(2) moieties derived from HEK293S GnTI(-) cells; (iv) insect cell-produced HA proteins carrying paucimannosidic N-glycans; and (v) HEK293S GnTI(-) cell-produced HA proteins treated with endoglycosidase H, thus carrying side chains composed of only a single N-acetylglucosamine each. The different HA glycosylation states were confirmed by comparative electrophoretic analysis and by mass spectrometric analysis of released glycans. The immunogenicity of the HA preparations was studied in chickens and mice. The results demonstrate that HA proteins carrying terminal mannose moieties induce significantly lower hemagglutination inhibition antibody titers than HA proteins carrying complex glycans or single N-acetylglucosamine side chains. However, the glycosylation state of the HA proteins did not affect the breadth of the antibody response as measured by an HA1 antigen microarray. We conclude that the glycosylation state of recombinant antigens is a factor of significant importance when developing glycoprotein-based vaccines, such as recombinant HA proteins.

Variants of the recently discovered avian gyrovirus 2 are detected in Southern Brazil and The Netherlands.

A genome of a virus preliminarily named avian gyrovirus 2 (AGV2), a close relative to chicken anemia virus, was recently discovered in a chicken in the state of Rio Grande do Sul, Southern Brazil. To study the occurrence of AGV2 in Rio Grande do Sul and the neighboring state Santa Catarina, a number of adult chickens (n=108 and n=48, respectively) were tested for the presence of AGV2 DNA. An AGV2-specific PCR was developed, optimized and used to analyze DNA extracted from clinical samples. AGV2 DNA was detected in 98/108 (90.7%) of samples collected in the state of Rio Grande do Sul and 29/48 (60.4%) of the samples collected in the state of Santa Catarina. In order to check whether AGV2 DNA would be detected in samples from a geographically distant region, DNA from brain samples of 21 diseased chickens from the Netherlands were tested independently, by the same method. In such specimens, 9/21 (42.9%) brain tissue samples were found to contain AVG2 DNA. Sequence analysis of some of the PCR products demonstrated that the amplified AGV2 sequences could vary up to 15.8% and could preliminarily be divided in three groups. This indicated the occurrence of variants of AGV2, which may reflect differences in geographical origin and/or in biological properties. The data presented here provides evidence that AGV2 seems fairly distributed in chickens in Southern Brazil and that AGV2 also circulates in the Netherlands. Besides, circulating viruses display genetic variants whose significance should be further examined, particularly to determine whether AGV2 would play any role in chicken diseases.

A highly conserved neutralizing epitope on group 2 influenza A viruses.

Current flu vaccines provide only limited coverage against seasonal strains of influenza viruses. The identification of V(H)1-69 antibodies that broadly neutralize almost all influenza A group 1 viruses constituted a breakthrough in the influenza field. Here, we report the isolation and characterization of a human monoclonal antibody CR8020 with broad neutralizing activity against most group 2 viruses, including H3N2 and H7N7, which cause severe human infection. The crystal structure of Fab CR8020 with the 1968 pandemic H3 hemagglutinin (HA) reveals a highly conserved epitope in the HA stalk distinct from the epitope recognized by the V(H)1-69 group 1 antibodies. Thus, a cocktail of two antibodies may be sufficient to neutralize most influenza A subtypes and, hence, enable development of a universal flu vaccine and broad-spectrum antibody therapies.

A single immunization with soluble recombinant trimeric hemagglutinin protects chickens against highly pathogenic avian influenza virus H5N1.

BACKGROUND: The highly pathogenic avian influenza (HPAI) virus H5N1 causes multi-organ disease and death in poultry, resulting in significant economic losses in the poultry industry. In addition, it poses a major public health threat as it can be transmitted directly from infected poultry to humans with very high (60%) mortality rate. Effective vaccination against HPAI H5N1 would protect commercial poultry and would thus provide an important control measure by reducing the likelihood of bird-to-bird and bird-to-human transmission. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we evaluated the vaccine potential of recombinant soluble trimeric subtype 5 hemagglutinin (sH5(3)) produced in mammalian cells. The secreted, purified sH5(3) was biologically active as demonstrated by its binding to ligands in a sialic acid-dependent manner. It was shown to protect chickens, in a dose-dependent manner, against a lethal challenge with H5N1 after a single vaccination. Protected animals did not shed challenge virus as determined by a quantitative RT-PCR on RNA isolated from trachea and cloaca swabs. Also in mice, vaccination with sH5(3) provided complete protection against challenge with HPAI H5N1. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that sH5(3) constitutes an attractive vaccine antigen for protection of chickens and mammals against HPAI H5N1. As these recombinant soluble hemagglutinin preparations can be produced with high yields and with relatively short lead time, they enable a rapid response to circulating and potentially pandemic influenza viruses.

Cytokine release in tissue-engineered epidermal equivalents after prolonged mechanical loading.

Prolonged mechanical loading of soft tissues may result in degeneration of these tissues, resulting in formation of pressure ulcers. The risk assessment of individuals might be improved by including measurements of the tissue response to mechanical loading. Cytokines, which are released by the top layer of the skin upon chemical irritation, might be used to determine the epidermal response to mechanical loading. This chapter describes methods to measure the release of cytokines IL-1 alpha, IL-1RA, and IL-8 from tissue-engineered epidermal equivalents in response to sustained mechanical loading. A custom-built loading device was used to apply load to the epidermal equivalents and the cytokines were measured using an enzyme-linked immunosorbent assay.

Pre- and postexposure use of human monoclonal antibody against H5N1 and H1N1 influenza virus in mice: viable alternative to oseltamivir.

New strategies to prevent and treat influenza virus infections are urgently needed. A recently discovered class of monoclonal antibodies (mAbs) neutralizing an unprecedented spectrum of influenza virus subtypes may have the potential for future use in humans. Here, we assess the efficacies of CR6261, which is representative of this novel class of mAbs, and oseltamivir in mice. We show that a single injection with 15 mg/kg CR6261 outperforms a 5-day course of treatment with oseltamivir (10 mg/kg/day) with respect to both prophylaxis and treatment of lethal H5N1 and H1N1 infections. These results justify further preclinical evaluation of broadly neutralizing mAbs against influenza virus for the prevention and treatment of influenza virus infections.

Challenges for porcine reproductive and respiratory syndrome virus (PRRSV) vaccinology.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a threat for the pig industry. Vaccines have been developed, but these failed to provide sustainable disease control, in particular against genetically unrelated strains. Here we give an overview of current knowledge and gaps in our knowledge that may be relevant for the development of a future generation of more effective vaccines. PRRSV replicates in cells of the monocyte/macrophage lineage, induces apoptosis and necrosis, interferes with the induction of a proinflammatory response, only slowly induces a specific antiviral response, and may cause persistent infections. The virus appears to use several evasion strategies to circumvent both innate and acquired immunity, including interference with antigen presentation, antibody-mediated enhancement, reduced cell surface expression of viral proteins, and shielding of neutralizing epitopes. In particular the downregulation of type I interferon-alpha production appears to interfere with the induction of acquired immunity. Current vaccines are ineffective because they suffer both from the immune evasion strategies of the virus and the antigenic heterogeneity of field strains. Future vaccines therefore must "uncouple" the immune evasion and apoptogenic/necrotic properties of the virus from its immunogenic properties, and they should induce a broad immune response covering the plasticity of its major antigenic sites. Alternatively, the composition of the vaccine should be changed regularly to reflect presently and locally circulating strains. Preferably new vaccines should also allow discriminating infected from vaccinated pigs to support a virus elimination strategy. Challenges in vaccine development are the incompletely known mechanisms of immune evasion and immunity, lack of knowledge of viral sequences that are responsible for the pathogenic and immunosuppressive properties of the virus, lack of knowledge of the forces that drive antigenic heterogeneity and its consequences for immunogenicity, and a viral genome that is relatively intolerant for subtle changes at functional sites.

The transport profile of cytokines in epidermal equivalents subjected to mechanical loading.

Pressure ulcer risk assessment might be optimized by incorporating the soft tissue reaction to mechanical loading in the currently used risk assessment scales. Cytokines, like IL-1alpha, IL-1RA, IL-8, and TNF-alpha, might be used to determine this tissue reaction, since they are released after 24 h of mechanical loading of epidermal equivalents. In the current study, the release and transport of these cytokines with time was evaluated. Epidermal equivalents were subjected to 20 kPa for different time periods (1, 2, 4, 6, 8, 16, and 24 h). Compared to the unloaded control group, a significant increase was found for IL-1alpha (4.7-fold), IL-1RA (4.8-fold), and IL-8 (3.6-fold) release after 1 h loading. For TNF-alpha, the release was significantly increased after 4 h of loading (5.1-fold compared to the unloaded situation), coinciding with the first signs of gross structural tissue damage. These cytokine values were determined in the surrounding medium and a transport model was developed to evaluate the distribution of cytokines inside the culture. These simulations revealed that all IL-8 and TNF-alpha was released from the keratinocytes, whereas most of the IL-1alpha and IL-1RA remained inside the keratinocytes during the 24 h loading period. In conclusion, IL-1alpha, IL-1RA, and IL-8 appear promising biochemical markers for pressure ulcer risk assessment, since their release is increased after 1 h of epidermal loading and before the onset of structural tissue damage.

Heterosubtypic neutralizing monoclonal antibodies cross-protective against H5N1 and H1N1 recovered from human IgM+ memory B cells.

BACKGROUND: The hemagglutinin (HA) glycoprotein is the principal target of protective humoral immune responses to influenza virus infections but such antibody responses only provide efficient protection against a narrow spectrum of HA antigenic variants within a given virus subtype. Avian influenza viruses such as H5N1 are currently panzootic and pose a pandemic threat. These viruses are antigenically diverse and protective strategies need to cross protect against diverse viral clades. Furthermore, there are 16 different HA subtypes and no certainty the next pandemic will be caused by an H5 subtype, thus it is important to develop prophylactic and therapeutic interventions that provide heterosubtypic protection. METHODS AND FINDINGS: Here we describe a panel of 13 monoclonal antibodies (mAbs) recovered from combinatorial display libraries that were constructed from human IgM(+) memory B cells of recent (seasonal) influenza vaccinees. The mAbs have broad heterosubtypic neutralizing activity against antigenically diverse H1, H2, H5, H6, H8 and H9 influenza subtypes. Restriction to variable heavy chain gene IGHV1-69 in the high affinity mAb panel was associated with binding to a conserved hydrophobic pocket in the stem domain of HA. The most potent antibody (CR6261) was protective in mice when given before and after lethal H5N1 or H1N1 challenge. CONCLUSIONS: The human monoclonal CR6261 described in this study could be developed for use as a broad spectrum agent for prophylaxis or treatment of human or avian influenza infections without prior strain characterization. Moreover, the CR6261 epitope could be applied in targeted vaccine strategies or in the design of novel antivirals. Finally our approach of screening the IgM(+) memory repertoire could be applied to identify conserved and functionally relevant targets on other rapidly evolving pathogens.

Diffusion measurements in epidermal tissues with fluorescent recovery after photobleaching.

BACKGROUND/PURPOSE: Pressure ulcers are areas of soft tissue breakdown, resulting from sustained mechanical loading of the skin and underlying tissues. Measuring biochemical markers that are released upon mechanical loading by the epidermis seems a promising method for objective risk assessment of the development of pressure ulcers. This risk assessment method will better determine the risk of a patient to develop pressure ulcers than the risk score lists currently used. So far, experimental studies have been performed that measure the tissue response in the culture supernatant. To elucidate the transport of the biochemical markers within the epidermis, the diffusion coefficient needs to be established. METHODS: In the current study, fluorescent recovery after photobleaching (FRAP) is used to determine the diffusion coefficient of fluorescent-labeled dextran molecules in human epidermis, porcine epidermis and engineered epidermal equivalents. These dextran molecules have a similar weight to the biochemical markers. RESULTS: Similar diffusion coefficients were found for human and porcine epidermal samples (6.2 x 10(-5)+/-1.2 x 10(-5) and 5.9 x 10(-5)+/-6.1 x 10(-6) mm2/s, respectively), whereas the diffusion coefficient of the engineered epidermal equivalent was significantly lower (2.3 x 10(-5)+/-1.0 x 10(-5) mm2/s). CONCLUSION: The diffusion could be measured in epidermal tissues using FRAP. In the future, the diffusion coefficients obtained in the current study will be used to study the difference between the transport in EpiDerm cultures and in human epidermis.

The nsp1alpha and nsp1 papain-like autoproteinases are essential for porcine reproductive and respiratory syndrome virus RNA synthesis.

The two N-terminal cleavage products, nsp1alpha and nsp1beta, of the replicase polyproteins of porcine reproductive and respiratory syndrome virus (PRRSV) each contain a papain-like autoproteinase domain, which have been named PCPalpha and PCPbeta, respectively. To assess their role in the PRRSV life cycle, substitutions and deletions of the presumed catalytic cysteine and histidine residues of PCPalpha and PCPbeta were introduced into a PRRSV infectious cDNA clone. Mutations that inactivated PCPalpha activity completely blocked subgenomic mRNA synthesis, but did not affect genome replication. In contrast, mutants in which PCPbeta activity was blocked proved to be non-viable and no sign of viral RNA synthesis could be detected, indicating that the correct processing of the nsp1beta/nsp2 cleavage site is essential for PRRSV genome replication. In conclusion, the data presented here show that a productive PRRSV life cycle depends on the correct processing of both the nsp1alpha/nsp1beta and nsp1beta/nsp2 junctions.

The free diffusion of macromolecules in tissue-engineered skeletal muscle subjected to large compression strains.

Pressure-related deep tissue injury (DTI) represents a severe pressure ulcer, which initiates in compressed muscle tissue overlying a bony prominence and progresses to more superficial tissues until penetrating the skin. Individual subjects with impaired motor and/or sensory capacities are at high risk of developing DTI. Impaired diffusion of critical metabolites in compressed muscle tissue may contribute to DTI, and impaired diffusion of tissue damage biomarkers may further impose a problem in developing early detection blood tests. We hypothesize that compression of muscle tissue between a bony prominence and a supporting surface locally influences the diffusion capacity of muscle. The objective of this study was therefore, to determine the effects of large compression strains on free diffusion in a tissue-engineered skeletal muscle model. Diffusion was measured with a range of fluorescently labeled dextran molecules (10, 20, 150kDa) whose sizes were representative of both hormones and damage biomarkers. We used fluorescence recovery after photobleaching (FRAP) to compare diffusion coefficients (D) of the different dextrans between the uncompressed and compressed (48-60% strain) states. In a separate experiment, we simulated the effects of local partial muscle ischemia in vivo, by reducing the temperature of compressed specimens from 37 to 34 degrees C. Compared to the D in the uncompressed model system, values in the compressed state were significantly reduced by 47+/-22% (p<0.02). A 3 degrees C temperature decrease further reduced D in the compressed specimens by 10+/-6% (p<0.05). In vivo, the effects of large strains and ischemia are likely to be summative, and hence, the present findings suggest an important role of impaired diffusion in the etiology of DTI, and should also be considered when developing biochemical screening methods for early detection of DTI.

Mechanisms that play a role in the maintenance of the calcium gradient in the epidermis.

BACKGROUND/PURPOSE: Calcium regulates the proliferation and differentiation of keratinocytes and plays a role in restoration of the epidermal barrier function. The factors that maintain the calcium gradient in vivo are still unknown. A numerical model may give more insight into transport processes that maintain the epidermal calcium gradient. METHODS: In this study, transport of free calcium in the epidermis is described with diffusion, convection and electrophoresis. Binding and release of calcium results in equilibrium between free and bound calcium. The physiological epidermal calcium gradient as well as the calcium concentration in a damaged epidermis are modeled. RESULTS: The typical shape of the calcium gradient in the epidermis, as found in experimental studies, was maintained when separate formulations were used for free and bound calcium. Application of damage results in a decrease of the calcium concentration, especially in the upper living epidermis. Using this model, an estimate could be made about the fraction bound calcium in the epidermis. CONCLUSION: The typical shape of the gradient is predominantly determined by the bound calcium concentration. For both a normal and a damaged epidermis, the concentration of free calcium is mainly determined by electrophoresis in the living epidermis, whereas in the largest part of the stratum corneum diffusion is the most important factor. The convection that was determined by the transepidermal water loss did not have an effect on the calcium concentration.