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Axolotls (Ambystoma mexicanum) may heal their skin wounds scar-free in both paedomorphs and metamorphs. In previous studies on small punch skin wounds, rapid re-epithelialisation was noted in these two axolotl morphs. However, large wound size in mammals may affect wound healing. In this study, large circumferential full thickness excision wounds on the hind limbs were created on juvenile paedomorphic and metamorphic axolotls. The results showed re-epithelialisation was more quickly initiated in paedomorphs than in metamorphs after wounding. The migrating rate of epidermis on the wound bed was faster in paedomorphs than in metamorphs and thus completion of re-epithelialisation was faster in paedomorphs than in metamorphs. Within these re-epithelialisation periods, neither basement membrane nor dermis was reformed. Epidermal cell proliferation was detected by EdU-labelling technique. In the normal unwounded skin, epidermal proliferation rate was higher in paedomorphs than in metamorphs. After wounding, the epidermal proliferation rate was significantly lower in the migrating front on the wound bed than in the normal skin in paedomorphs. The EdU-labelling rate between normal skin and migration front was not different in metamorphs. Lacking of more proliferating epidermal cells on the wound bed indicated that the new epidermis here derived rather from migrating epidermal cells than from cell proliferation in situ. In conclusion, re-epithelialisation in the large wound might be fully completed in both morphs despite it was initiated earlier and with faster rate in paedomorphs than in metamorphs. The new epidermis on the wound bed derived mainly from cell migration than by cell proliferation in the re-epithelialisation period. J. Morphol. 278:228-235, 2017. © 2016 Wiley Periodicals,Inc.

Among the cytochrome P 450 (CYP) enzymes, CYP3A4 is most abundantly existed and metabolizes most of the drugs. We have shown human dermal fibroblasts (HDFs) express low levels of CYPs, but could be engineered to express much higher levels to be used as drug metabolism prediction. To seek better cell source for engineering, the basal mRNA expression and enzyme activities of CYP3A4 in HDFs ( n = 5), bone marrow-derived mesenchymal stem cells (BMMSCs, n = 7), and adipose-derived stem cells (ADSCs, n = 3) were compared. The basal CYP3A4 expression and activity in BMMSCs and ADSCs were significantly higher than in HDFs. However, the coefficient of variation of CYP3A4 expression was very high in each cell type. Comparing the expression levels of 4 regulators glucocorticoid receptor, pregnane X receptor ( PXR ), hepatocyte nuclear factor 4α, and hydrocarbon receptor nuclear translocator in the total 15 cell samples, we found the only basal PXR expression was significantly correlated with CYP3A4 expression. The gender and age of patients were not significant determinants in the expressions of PXR and CYP3A4 . In conclusion, BMMSCs and ADSCs expressed higher levels of CYP3A4 than HDFs and the expression of CYP3A4 might be mainly determined by basal PXR expression.

Parvovirus 4 (PARV4) is one of the emerging human parvoviruses discovered recently. PARV4 has long-lasting persistence in tissues after primary infection, but persistent PARV4 viremia has yet to be effectively detected in humans. In the present work, longitudinal serum samples from eleven patients with antiphospholipid syndrome were tested by nested polymerase chain reaction for the presence of PARV4 DNA. In one patient, PARV4 4 DNA was detected in all longitudinal serum samples collected over a period of 119 months. In addition, PARV4 DNA was present in two or more longitudinal serum samples from seven other patients. Two possible explanations are a persistent infection with intermittent low viral load below detection limit and a recurrent reactivation of latent infection. In conclusion, to our knowledge, this is the first direct evidence of detection of persistent PARV4 infection. Placental transmission may be one of the major routes of PARV4 infection in endemic areas if women of child-bearing age have continuous or intermittent circulating PARV4 DNA seen in our patients.

Background/Purpose We evaluated the long-term effects of green tea extract (GTE) supplementation on oxidative stress, biliary acute phase protein expression, and liver function in CCl4-induced chronic liver injury. Methods We evaluated the antioxidant activity of GTE in comparison with those of vitamin C, vitamin E, and β-carotene in vitro by using an ultrasensitive chemiluminescence analyzer. Chronic liver injury was induced by intraperitoneally administering carbon tetrachloride (CCl4) (1 mL/kg body weight, twice weekly) to female Wistar rats for 8 weeks. The effects of low (4 mg/kg body weight per day) and high (20 mg/kg body weight per day) doses of intragastric GTE on CCl4-induced liver dysfunction and fibrosis were examined by measuring the bile and blood reactive oxygen species levels and biochemical parameters by using Western blot and two-dimensional polyacrylamide gel electrophoresis techniques. Results GTE has greater scavenging activity against O2–, H2O2, and Hypochlorous acid (HOCl) in vitro than vitamin C, vitamin E, and β-carotene do. In vivo, CCl4 markedly increased bile and blood reactive oxygen species production, lipid accumulation, number of infiltrated leukocytes, fibrosis, hepatic hydroxyproline content, and plasma alanine aminotransferase and aspartate aminotransferase activities, and reduced plasma albumin levels. Two-dimensional polyacrylamide gel electrophoresis revealed that CCl4 increased the acute-phase expression of six biliary proteins and decreased hepatic B-cell lymphoma 2 (Bcl-2), catalase, and CuZn superoxide dismutase protein expression. GTE supplementation attenuated CCl4-enhanced oxidative stress, levels of biochemical parameters, pathology, and acute-phase protein secretion, and preserved antioxidant/antiapoptotic protein expression. Conclusion GTE supplementation attenuates CCl4-induced hepatic oxidative stress, fibrosis, acute phase protein excretion, and hepatic dysfunction via the antioxidant and antiapoptotic defense mechanisms.

The Penicillium genus of fungi is a frequently reported cause of allergic reactions. However, only a limited number of allergens have been reported. In Penicillium spp., many allergens show higher IgE-binding activity in culture filtrate extracts than in cellular extracts. In order to investigate the IgE-reactive profile of mold-sensitized patients, secreted IgE-reactive proteins from Penicillium citrinum were identified by 2-DE, serum immunoblotting, and nanoLC-MS/MS. Among the IgE-reactive spots, one known allergen, Pen c 13, and four novel allergens were identified. The cDNAs coding for Pen c 32 and Pen c 30 were cloned using designed primers based on nanoLC-MS/MS analysis. The amino acid sequences of Pen c 32 and Pen c 30 were, respectively, found to have extensive similarity with those of pectate lyases and catalases from various fungi. Native Pen c 30 was shown to have catalase activity and to bind to serum IgE from 48% of mold-allergic patients and induced immediate type skin reactions in a sensitized patient. Here, we present a proteome approach which resulted in the identification of four novel secreted allergens. These novel allergens might be useful in allergy diagnosis and in the treatment of mold-allergic disorders.

Anti-CD45 antibody exhibits multiple biological effects on human mononuclear cells (MNC) and polymorphonuclear neutrophils (PMN). We intended to determine whether anti-CD45 antibody could affect arachidonic acid metabolism and thereby, the interactions between human naïve MNC and PMN.Human naïve MNC and PMN were incubated with monoclonal anti-human CD45 IgG F(ab')(2) antibody or non-specific IgG F(ab')(2) for 30 min. The mRNA expression of cyclooxygenase type 1 (COX-1), type 2 (COX-2), 5-lipoxygenase (5-LOX) and leukotriene A(4) hydrolase (LTA(4) hydrolase) in both cells was detected by RT-PCR and quantified by densitometric determination. The presence of COX-1 and COX-2 molecules in the cells was detected by Western blot. The concentration of PGE(2) and LTB(4) in cultured supernatants was measured by EIA kits.Anti-CD45 IgG F(ab')(2) up-regulated LTA(4) hydrolase mRNA expression and LTB(4) production, but down-regulated COX-1 and COX-2 mRNA expression and PGE2 production, of naïve MNC compared to non-specific IgG F(ab')(2). In contrast, a reverse modulation by the specific antibody on PMN was observed including up-regulation of cyclooxygenase pathway and down-regulation of lipoxygenase pathway.A novel activity of anti-CD45 with reverse modulation on cyclooxygenase/lipoxygenase pathways was found such that the expression of COX-1 and COX-2 in PMN, and 5-LOX and LTA(4) hydrolase in MNC were enhanced.

It is generally thought that parvovirus B19 is stable genetically. Consistently, genetic drift has not been found in patients with persistent B19 infection. In this report, longitudinal genetic changes in NS1 and VP1 gene of B19 isolates from three AIDS patients with persistent B19 infection were studied. One of the three patients was not treated with highly active anti-retroviral therapy (HAART). B19 viral DNA from these patients was amplified by polymerase chain reaction (PCR) and then sequenced directly. A single genetic change was found in the B19 isolate obtained from the patient not treated with HAART on Day 10 after intravenous immunoglobulin (IVIG) treatment. The nucleotide sequences of B19 isolated from this patient, then remained unchanged over a period of 11 months. Analysis of NS1 clones derived from his longitudinal viral isolates showed the existence of quasi-species but genetic drift was not found. One of the other two patients treated with HAART experienced treatment failure; he was later treated with mega-HAART. In contrast to the genetic stability of B19 isolates from the patient not treated with HAART, multiple genetic changes were discovered in the viral isolates from the two other patients after HAART and mega-HAART, respectively. Through analysis of B19 clones, the frequency of clones containing these mutations confirmed the genetic drift. Nucleotide substitutions seen in VP2 gene of isolates with genetic drift from both patients were all non-conserved, suggesting that they are positively selected.

Background The transmission routes of PARV4 are not completely understood. The first PARV4 serological study suggested that PARV4 is transmitted predominantly through parenteral route. Objectives To set up an immunoblot system for studying the epidemiology of PARV4 infection in HIV-1 infected patients in Taiwan. Study design Recombinant fusion proteins SUMOVP2 (a.a. 272–630 of PARV4 open reading frame 2) and SUMOVP3 (a.a. 604–914) were made and used as antigens in immunoblot. Plasma samples were from HIV-1 infected intravenous drug users IDU (69), homosexuals (66) and heterosexuals (68). Results PARV4 IgG seropositive rate was 73.9%, 71.2% and 58.8%; IgM seropositive rate was 40.5%, 16.7% and 17.6% in IDUs, homosexuals and heterosexuals, respectively. Longitudinal samples were available from two homosexuals positive for IgM anti-PARV4, persistent IgM response was found over a period of 9 and 21 months, respectively. Conclusions PARV4 is a common viral infection in HIV-1 infected homosexuals and heterosexuals in Taiwan. The detection of IgM anti-PARV4 does not always suggest recent PARV4 infection.

Anaemia caused by persistent parvovirus B19 infection can be remitted by intravenous immunoglobulin (IVIG) treatment. However, maintenance IVIG therapy is required in some AIDS patients as well as patients with combined immuno-deficiency because of relapse of anaemia. We used a simple semi-quantitative polymerase chain reaction (PCR) method to trace plasma B19 viral load after administration of IVIG. We noted that the rebound of B19 viral load occurred 6 weeks prior to detection of anaemia. This method may be helpful in determining the interval of maintenance IVIG therapy.

Background Pen c 13, identified as a 33-kDa alkaline serine protease, is a major allergen secreted by Penicillium citrinum. Detailed knowledge about the epitopes responsible for IgE binding would help inform the diagnosis/prognosis of fungal allergy and facilitate the rational design of hypoallergenic candidate vaccines. The goal of the present study was to characterize the IgE epitopes of Pen c 13. Methodology/Principal Findings Serum samples were collected from 10 patients with mold allergy and positive Pen c 13 skin test results. IgE-binding epitopes on rPen c 13 were mapped using an enzymatic digestion and chemical cleavage method, followed by dot-blotting and mass spectrometry. A B-cell epitope-predicting server and molecular modeling were used to predict the residues most likely involved in IgE binding. Theoretically predicted IgE-binding regions were further confirmed by site-directed mutagenesis assays. At least twelve different IgE-binding epitopes located throughout Pen c 13 were identified. Of these, peptides S16 (A148–E166) and S22 (A243–K274) were recognized by sera from 90% and 100% of the patients tested, and were further confirmed by inhibition assays. Peptide S22 was selected for further analysis of IgE-binding ability. The results of serum screening showed that the majority of IgE-binding ability resided in the C-terminus. One Pen c 13 mutant, G270A (T261–K274), exhibited clearly enhanced IgE reactivity, whereas another, K274A, exhibited dramatically reduced IgE reactivity. Conclusions/Significance Experimental analyses confirmed in silico-predicted residues involved in an important antigenic region of Pen c 13. The G270A mutant of Pen c 13 has the potential to serve as an additional tool for the diagnosis/prognosis of mold allergy, and the K274A mutant, as a hypoallergenic form of the epitope, may provide a framework for the design and development of a safe and efficient therapeutic strategy for treating human allergic diseases.

Background The transmission routes for human parvovirus 4 (PARV4) infections in areas with high seroprevalence are not known. In the work described here, persistent PARV4 viral replication was investigated by conducting a longitudinal study. Methods Ten healthcare workers each provided a blood sample at the beginning of the study (first sample) and 12 months later (second sample). The paired samples were tested for PARV4-positivity by immunoblotting analysis and nested polymerase chain reactions. Results IgG antibodies against PARV4 were detected in six participants, three of whom also had IgM antibodies against PARV4. The immunoblotting results did not vary over time. PARV4 DNA was detected in the first blood sample from one participant who had IgG antibodies against PARV4 and in the second blood samples from 2 participants who had IgG and IgM antibodies against PARV4. Conclusions Detection of PARV4 DNA in the second blood samples from two seropositive participants suggests the existence of persistent PARV4 replication or reactivation of inactive virus in the tissues. The finding of persistent or intermittent PARV4 replication in individuals with past infections provides an important clue toward unraveling the non-parenteral transmission routes of PARV4 infection in areas where the virus is endemic.