Effect of amino acid variations in the central region of human serum amyloid A on the amyloidogenic properties.

Human serum amyloid A (SAA) is a precursor protein of the amyloid fibrils that are responsible for AA amyloidosis. Of the four human SAA genotypes, SAA1 is most commonly associated with AA amyloidosis. Furthermore, SAA1 has three major isoforms (SAA1.1, 1.3, and 1.5) that differ by single amino acid variations at two sites in their 104-amino acid sequences. In the present study, we examined the effect of amino acid variations in human SAA1 isoforms on the amyloidogenic properties. All SAA1 isoforms adopted α-helix structures at 4°C, but were unstructured at 37°C. Heparin-induced amyloid fibril formation of SAA1 was observed at 37°C, as evidenced by the increased thioflavin T (ThT) fluorescence and ß-sheet structure formation. Despite a comparable increase in ThT fluorescence, SAA1 molecules retained their α-helix structures at 4°C. At both temperatures, no essential differences in ThT fluorescence and secondary structures were observed among the SAA1 isoforms. However, the fibril morphologies appeared to differ; SAA1.1 formed long and curly fibrils, whereas SAA1.3 formed thin and straight fibrils. The peptides corresponding to the central regions of the SAA1 isoforms containing amino acid variations showed distinct amyloidogenicities, reflecting their direct effects on amyloid fibril formation. These findings may provide novel insights into the influence of amino acid variations in human SAA on the pathogenesis of AA amyloidosis.

Factitious purpura in a 10-year-old girl.

We describe a 10-year-old girl who presented with bizarre purpura. Both congenital and autoimmune hemorrhagic disorders were excluded based on her past medical history and physical and laboratory findings. Child abuse was also ruled out as purpura continued to develop after child-family separation. Histologic examination of the skin lesions revealed disruption of collagen fiber bundles. This finding indicated application of external force, leading to a definitive diagnosis of factitious purpura. Although it is very rare in school-age children, the diagnosis of factitious purpura should be included in the differential diagnosis of purpura in children. Histologic analysis of skin biopsies may aid in establishing the diagnosis.

Adjuvant effect of lipopolysaccharide on the induction of contact hypersensitivity to haptens in mice.

BACKGROUND: Toll-like receptor (TLR) 4 is a critical receptor and signal transducer for lipopolysaccharide (LPS), a major component of Gram-negative bacteria. The MyD88-independent pathway downstream of TLR4 leads to functional dendritic cell (DC) maturation, although LPS-induced cytokine production from DCs is MyD88-dependent. OBJECTIVES: We investigated whether intracutaneously injected LPS alters the functions of cutaneous DCs, leading to enhanced contact hypersensitivity (CH). METHODS: The ear swelling response was measured to evaluate the magnitude of CH. Cell proliferation of allogeneic splenocytes stimulated by DC-enriched draining lymph node (LN) cells was measured by performing a [(3)H]-thymidine incorporation assay. Epidermal I-A+ cells were evaluated under an epifluorescent microscope. I-A+ FITC-bearing cells from the draining LNs 24h after FITC application were analyzed on FACScan. RESULTS: LPS augmented CH induction in C3H/HeN (HeN) and MyD88-knockout (KO) mice but not in C3H/HeJ (HeJ) and H-2S(d)-bearing strains such as BALB/c mice. LPS failed to augment the allo-stimulatory ability of DCs in the draining LNs after hapten applications. LPS altered the density and morphology of epidermal I-A+ cell in HeN and BALB/c mice but not in TLR4-deficient HeJ mice. LPS increased the proportion of I-A+ FITC-bearing cells in the LNs 24h after FITC application in HeN, but not in BALB/c and HeJ. CONCLUSIONS: LPS augments the ability of DCs to migrate to the draining LNs, leading to enhanced CH via a TLR4-dependent, MyD88-independent pathway. The different effects of LPS on CH in some strains of mice may explain individual differences in the susceptibility to establish CH to daily antigen exposures in clinical settings.

Regulatory effects of antihistamines on the responses to staphylococcal enterotoxin B of human monocyte-derived dendritic cells and CD4+ T cells.

BACKGROUND: Antihistamines are widely used for the treatment of allergic diseases, such as urticaria and allergic rhinitis. They are also effective for the treatment of diseases in which T cells are mainly involved in the pathogenesis, such as atopic dermatitis (AD) and contact dermatitis. Dendritic cells (DCs) drive polarization of naive T cells into Th1 or Th2 subsets, and are also likely to be involved in AD pathogenesis. OBJECTIVES: The aim of this study was to determine the effects of antihistamines on DCs and CD4+ T cells. METHODS: Human monocyte-derived DCs (MoDCs) and autologous CD4+ T cells were obtained from healthy subjects, and cultured together or independently in the presence of antihistamines. As a stimulant, we used staphylococcal enterotoxin B or the combination of anti-CD3 monoclonal antibody (mAb) and anti-CD28 mAb. The concentrations of cytokines and chemokines in culture supernatants were measured by ELISA. The expression of surface molecules on MoDCs was measured by flow cytometry. Cell proliferation in the cocultures of MoDCs and CD4+ T cells (DC-T cocultures) was measured by a [(3)H] thymidine incorporation assay. RESULTS: Antihistamines inhibited the production of IFN-gamma, and enhanced the production of IL-4 in DC-T cocultures. Antihistamines inhibited the production of TNF-alpha, TARC, MDC, IP-10, and Mig from MoDCs. Epinastine, one of antihistamines, suppressed the expression of ICAM-1 (CD54) on MoDCs. Epinastine also inhibited the proliferation of CD4+ T cells cocultured with MoDCs. CONCLUSIONS: Our findings show that antihistamines regulate immune responses by affecting the interaction between DCs and CD4+ T cells, and further DCs and CD4+ T cells independently, which may partially contribute to the control of allergic diseases such as AD and contact dermatitis.

Relationship among human herpesvirus 6 reactivation, serum interleukin 10 levels, and rash/graft-versus-host disease after allogeneic stem cell transplantation.

BACKGROUND: The relationship between herpesvirus reactivation and graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (allo-SCT) is unclear. OBJECTIVE: We sought to examine the relationship between human herpesvirus (HHV) reactivation and rash/GVHD after allo-SCT by prospective evaluation. METHODS: Fifteen patients who had received allo-SCT underwent prospective serial examinations for human herpesvirus 6 (HHV-6), HHV-7, cytomegalovirus, and Epstein-Barr virus DNA in the blood by polymerase chain reaction and real-time polymerase chain reaction. Serum interferon gamma, interleukins 4 and 10, tumor necrosis factor alpha, and soluble interleukin 2 receptor (sIL-2R) were also measured. RESULTS: In 10 of 15 patients, macular/papular eruptions were seen after allo-SCT and GVHD was diagnosed. In 8 patients with rash, HHV-6 DNA levels correlated with the cutaneous manifestation. Interleukin 10 and sIL-2R also increased in association with rash. LIMITATIONS: The number of patients in our study was relatively small. Not all patients were examined for cytokines and sIL-2R. CONCLUSIONS: HHV-6 reactivation may be involved in the pathogenesis of rash/GVHD after allo-SCT.

Paraneoplastic pemphigus presenting as erythrodermic lichenoid dermatitis with concomitant features of pemphigus foliaceus.

We describe a patient with paraneoplastic pemphigus who presented with erythrodermic lichenoid dermatitis, later developing blisters of pemphigus foliaceus type and oral erosive lesions. In addition to antibodies against the plakin family proteins, the patient's serum was positive for anti-desmoglein 1 antibodies without coexisting anti-desmoglein 3 activities by enzyme-linked immunosorbent assay, which is a very rare autoantibody profile in paraneoplastic pemphigus.

Role for connexin 26 in metastasis of human malignant melanoma: communication between melanoma and endothelial cells via connexin 26.

BACKGROUND: Connexins form the intercellular channels of the gap junction and play an integral part in a variety of biological functions, such as maintaining tissue homeostasis, cell growth control, and development. Previously it was demonstrated that the expression of connexin 26 (Cx26) can increase the metastatic potential of mouse melanoma cells. The objective of the study was to investigate the role Cx26 plays in the metastasis of human melanoma cells, focusing on the communication between melanoma cells and endothelial cells. METHODS: Immunostaining was used to examine Cx26 expression in the melanoma lesions and in the endothelial cells around the melanoma cell nests. In all, 33 melanomatous tissue samples from 16 patients were studied, as well as nevocellular nevus (NCN) and normal skin samples. Cx26 mRNA and protein expression was also examined in the cultured endothelial cells. A dye-transfer assay was performed to examine gap junction formation between melanoma cells and endothelial cells. RESULTS: Immunohistochemistry demonstrated that Cx26 was clearly expressed by the endothelial cells of the small vessels surrounding the melanoma cell nests as well as the melanoma cells. Cx26 mRNA and protein expression was detected in cultured endothelial cells. In a coculture system with human malignant melanoma cell line (HMY-1) and endothelial cells (HMVEC), immunohistochemistry indicated Cx26 expression in both types of cells and dye-transfer assay demonstrated dye-coupling from HMY-1 into HMVEC. CONCLUSIONS: Cx26 may contribute to the metastasis of melanoma by facilitating communication between melanoma cells and their surrounding endothelial cells.

Juvenile-onset hypergammaglobulinemic purpura and fetal congenital heart block.

Waldenström's hypergammaglobulinemic purpura (HGP) is a rare chronic disorder characterized by recurrent purpura on the legs, a polyclonal increase in serum gamma-globulin, an elevated erythrocyte sedimentation rate and a positive rheumatoid factor. A 30-year-old primigravid woman with 14 years of HGP was found to have fetal bradycardia at 25 weeks' gestation. Laboratory investigations demonstrated positive anti-Ro/SSA and anti-La/SSB antibodies in the maternal serum. Cesarean delivery was performed at 39 weeks, and a 2750-g female infant was born with complete atrioventricular block. Fortunately, the neonatal period has been uneventful without need for pace-making. Maternal HGP exacerbated just after delivery, but resolved within 1 week without treatment. Physicians should be aware of the possible presence of neonatal lupus-related anti-Ro/SSA and anti-La/SSB autoantibodies in patients with HGP. Screening for these autoantibodies is important and could be used as a marker to identify and manage high-risk pregnancies.

Compound heterozygous mutations including a de novo missense mutation in ABCA12 led to a case of harlequin ichthyosis with moderate clinical severity.

Harlequin ichthyosis (HI) is one of the most devastating genodermatoses. Recently, ABCA12 mutations were identified as the cause of HI. A newborn Japanese male demonstrated the typical features of HI. The patient was treated with oral etretinate and his general condition has been good (now aged 1.5 years). This patient with moderate clinical severity was compound heterozygous for a novel de novo missense mutation 1160G > A (S387N) in exon 10 and a maternal deletion mutation 4158_4160delTAC (T1387del) in exon 28 of ABCA12. T1387del was a deletion of a highly conserved threonine residue within the first adenosine 5' triphosphate-binding domain and is thought to seriously affect the function of the ABCA12 protein. Conversely, the residue 387 is located outside the known active sites of ABCA12 and S387N is predicted not to lead to a serious functional deficiency in ABCA12. Electron microscopy revealed abnormal lamellar granules in the granular layer cells and a moderate number of lipid vacuoles in the cornified cells. Disturbed glucosylceramide transport was confirmed in the cultured keratinocytes from the patient. No de novo mutation in ABCA12 has yet been reported either in HI or lamellar ichthyosis. The present case suggested that a de novo ABCA12 mutation might underlie HI.

Mosquito salivary gland extracts induce EBV-infected NK cell oncogenesis via CD4 T cells in patients with hypersensitivity to mosquito bites.

Severe hypersensitivity to mosquito bites (HMB) is characterized by intense local skin reactions and systemic symptoms such as high fever, lymphadenopathy, and hepatosplenomegaly. Patients with HMB often have natural killer (NK) cell lymphocytosis associated with Epstein-Barr virus (EBV) infection. Here we investigated whether mosquito bites have any influence on the oncogenesis of EBV-infected NK cells. We examined six HMB patients with EBV-infected NK cell lymphocytosis. We first demonstrated that CD4+ T cells, but not NK cells, proliferated well in response to mosquito salivary gland extracts (SGE), especially to SGE of Aedes albopictus. When NK cells were cocultured with autologous CD4+ T cells stimulated by mosquito SGE, the expression of viral oncogene latent membrane protein 1 (LMP1) was remarkably enhanced. Next, we stimulated mononuclear cells of the patients with mosquito SGE, and NK cell counts were monitored for 28 d. The counts changed little from initial levels in the culture with mosquito SGE, whereas they decreased steadily in the culture without the extracts. Furthermore, we detected LMP1 mRNA in the skin lesion induced by mosquito SGE. These results suggest that mosquito bites can induce expression of the viral oncogene LMP1 in NK cells via mosquito antigen-specific CD4+ T cells, which is involved in the oncogenesis of NK cells in vivo.

Bucillamine-induced toxic epidermal necrolysis and fixed drug eruption.

We report two cases of bucillamine-induced bullous reactions with keratinocyte necrosis. The first patient, a 27-year-old woman, developed toxic epidermal necrolysis (TEN) over her whole body after taking bucillamine 300 mg/day for seven days. The second patient, a 63-year-old woman, developed several bullous erythemas on the mucous membranes and legs after taking bucillamine for more than two years. The fixed drug eruptions were diagnosed based on a provocation test in addition to clinical and histopathologic findings. These cases highlight the importance of considering fixed drug eruption as well as TEN in the differential diagnosis of bucillamine-induced bullous drug eruption.

Fas gene mutations in mycosis fungoides: analysis of laser capture-microdissected specimens from cutaneous lesions.

Fas (APO-1/CD95) is a transmembrane protein which mediates programmed cell death (apoptosis). Cells with a mutated Fas gene are resistant to apoptosis and thus accumulate in lesional tissues. This might provide a basis for the development of neoplasias. Genomic DNA selectively obtained from Pautrier's microabscesses in 16 cases of mycosis fungoides (MF) using a laser capture microdissection method was analyzed. Fas gene mutations were detected in 3 of 16 cases of MF (18.8%); 1 was silent and 2 were missense mutations located in exon 9. One of the 2 missense mutations involved the death domain of the Fas gene, which is essential for apoptotic signal transduction. The missense mutations resulted in the substitution of Ala with Asp at codon 220 and Ile with Thr at codon 314. Mouse T cell lymphoma cells transfected with mutant genes were resistant to apoptosis induced by the anti-Fas antibody, indicating that the missense mutations found in MF were loss-of-function mutations, thus causing the accumulation of cells in the cutaneous lesions. These findings suggest that the accumulation of lymphoid cells with Fas mutations provides, in part, a basis for the development or maintenance of MF.

In situ detection of acetylaminofluorene-DNA adducts in human cells using monoclonal antibodies.

The present study was performed to generate monoclonal antibodies capable of detecting N-acetoxy-2-acetylaminofluorene (NA-AAF)-derived DNA adducts in human cells in situ. As an immunogen, we employed NA-AAF-modified single-stranded DNA coupled electrostatically to methylated protein and we produced five different monoclonal antibodies. All of them showed strong binding to NA-AAF-modified DNA, but had undetectable or minimal binding to undamaged DNA. Competitive inhibition experiments revealed that the epitope recognized by these antibodies is N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) in DNA, although deacetylated N-(deoxyguanosin-8-yl)-2-aminofluorene in DNA is also recognized with slightly less efficiency. In contrast, these antibodies did not bind to 3-(deoxyguanosin-N(2)-yl)-2-acetylaminofluorene in DNA or to UV-induced lesions in DNA. Interestingly, they showed only minimal binding to small AAF-nucleoside adducts (dG-C8-AAF), indicating that DNA regions flanking a DNA-bound adduct, in addition to the adduct itself, are essential for the stable binding of the antibodies. Using an enzyme-linked immunosorbent assay with the most promising antibody (AAF-1), we detected the concentration-dependent induction of NA-AAF-modified adducts in DNA from repair deficient xeroderma pigmentosum (XP) cells treated with physiological concentrations of NA-AAF. Moreover, the assay enabled to confirm that normal human cells efficiently repaired NA-AAF-induced DNA adducts but not XP-A cells. Most importantly, the formation of NA-AAF-induced DNA adducts in individual nuclei of XP cells could be clearly visualized using indirect immunofluorescence. Thus, we succeeded in establishing novel monoclonal antibodies capable of the in situ detection of NA-AAF-induced DNA adducts in human cells.

Trichothiodystrophy fibroblasts are deficient in the repair of ultraviolet-induced cyclobutane pyrimidine dimers and (6-4)photoproducts.

A photosensitive form of trichothiodystrophy (TTD) results from mutations in the same XPD gene as the DNA-repair-deficient genetic disorder xeroderma pigmentosum group D (XP-D). Nevertheless, unlike XP, no increase in skin cancers appears in patients with TTD. Although the ability to repair ultraviolet (UV)-induced DNA damage has been examined to explain their cancer-free phenotype, the information accumulated to date is contradictory. In this study, we determined the repair kinetics of cyclobutane pyrimidine dimers (CPD) and (6-4)photoproducts (6-4PP) in three TTD cell strains using an enzyme-linked immunosorbent assay. We found that all three TTD cell strains are deficient in the repair of CPD and of 6-4PP. UV sensitivity correlated well with the severity of repair defects. Moreover, accumulation of repair proteins (XPB and proliferating cell nuclear antigen) at localized DNA damage sites, detected using micropore UV irradiation combined with fluorescent antibody labeling, reflected their DNA repair activity. Importantly, mutations of the XPD gene affected both the recruitment of the TFIIH complex to DNA damage sites and the TFIIH expression. Our results suggest that there is no major difference in the repair defect between TTD and XP-D and that the cancer-free phenotype in TTD is unrelated to a DNA repair defect.

Expression of p53 family in scars.

BACKGROUND: There have been some reports on the relationship between p53 and keloid formation. However, there have been no studies comparing the p53 expression among scars in various stages of maturity. However, p63 and p73 have been identified as p53-related genes and have been found to be similar to p53 in their structures and functions and these proteins have also been suggested to relate to scar formation. OBJECTIVE: We investigate the expression of three proteins of the p53 family in scars with various clinical manifestations and discuss the shared features and differences of these proteins. METHODS: Forty untreated scar lesions consisting of keloids, hypertrophic scars, and atrophic scars were prepared for investigation. We detected the expression of p53, p63 and p73 proteins using Western blot analysis and histopathological study in each sample. RESULTS: The 40 lesions were divided into four groups according to their clinical manifestations: keloid (Group A), red hypertrophic scar (Group B), white and hard hypertrophic scar (Group C), atrophic white scar (Group D). In Groups A and B, the histopathological findings demonstrated increased fibroblasts, capillary vessels and infiltration of inflammatory cells. In Group C, most of these changes decreased but proliferation of collagen fibers was evident. In Group D, the degree of proliferation of collagen fibers was much less and capillary vessels and infiltration of inflammatory cells were not evident. The levels of p53 protein elevated in Groups A, B and C and were higher in order of Groups A, B and C. In Group D, the level of p53 was almost the same as that of the control. The level of p63 protein was almost the same as that of the control in all groups. The level of p73 protein was elevated only in Group C. CONCLUSION: The p53 family members behave in a different manner in various scar tissues. It is suggested that these proteins play different roles in scar formation and the development of unfavorable scars.

A case of multiple cavernous hemangiomas with skin and central nervous system involvement.

A 47-year-old woman presented with multiple bluish subcutaneous nodules on the trunk and upper extremities. The histological diagnosis of a subcutaneous nodule was cavernous hemangioma. Multiple cavernous hemangiomas were also found in her cerebrum, cerebellum, and medulla oblongata on magnetic resonance imaging examination. We did not detect any mutations in the two loci of the TIE2 gene that have been reported in familial venous malformations.

CD4+ T-lymphocyte-induced Epstein-Barr virus reactivation in a patient with severe hypersensitivity to mosquito bites and Epstein-Barr virus-infected NK cell lymphocytosis.

BACKGROUND: Natural killer (NK) cell lymphocytosis associated with Epstein-Barr virus (EBV) infection often shows severe hypersensitivity to mosquito bites (HMB) characterized by intense local skin reactions and systemic symptoms such as high fever, lymphadenopathy, and hepatosplenomegaly. However, the induction mechanism of HMB is still unclear. OBSERVATIONS: We investigated a typical case of HMB with EBV-positive NK cell lymphocytosis. CD4+ T cells dominantly infiltrated the site of the mosquito bite, while EBV-positive cells were few in comparison. CD4+ T cells, but not CD8+ T cells or NK cells, responded to the mosquito salivary gland extracts. Interestingly, coculturing of the NK cells and CD4+ T cells activated by mosquito extracts induced expression of EBV lytic-cycle proteins in the NK cells. Furthermore, the expression of BZLF1, a viral lytic-cycle transactivator, was detectable at the skin lesion induced by scratch patch testing with mosquito extract. The EBV DNA copy number levels in the plasma were elevated in systemic HMB symptoms compared with the normal condition. CONCLUSIONS: CD4+ T cells are important for the primary skin reaction to mosquito bites and might play a key role in reactivation of latent EBV infection in NK cells. This viral reactivation contributed to the pathogenesis of the infectious mononucleosis-like systemic symptoms of HMB in our present case.