Occult hepatitis B infections among blood donors in Lao PDR.

BACKGROUND AND OBJECTIVES: In Lao People's Democratic Republic, hepatitis B virus is highly endemic. However, blood donations are only screened for HBsAg, leaving a risk of transmission by HBsAg-negative occult infected donors. Here, we characterized first-time blood donors to assess prevalence of hepatitis B virus infections and occult infected donors. MATERIALS AND METHODS: Sera were screened for HBsAg, HBeAg and anti-HBs, anti-HBc and anti-HBe antibodies. Occult HBV infections (OBIs) were assessed in HBsAg-negative sera by PCR, and sera of HBsAg positive and occult infected donors were phylogenetically characterized. RESULTS: 9·6% of the donors were HBsAg positive, and 45.5% were positive for at least one of the hepatitis B virus serum markers. More than 40% HBsAg carriers were HBeAg positive, with HBeAg seroconversion occurring around 30 years of age. Furthermore, 10·9% of HBsAg-negative, anti-HBc and/or anti-HBs-positive donors were occult infected with hepatitis B virus. Thus, at least 3·9% of blood donations would potentially be unsafe, but hepatitis B virus DNA copy numbers greatly varied between donors. CONCLUSION: In Lao People's Democratic Republic, a sizable proportion of HBsAg-negative and anti-HBc antibody-positive blood donations are potentially DNA positive and infective for hepatitis B.

Detection of Human papillomavirus and cellular regulators p16INK4a, p53, and NF-kappaB in penile cancer cases in Kenya.

Human papillomavirus (HPV) E6 and E7 gene products play a central role in the induction of benign proliferation and malignant transformation by interacting with several cellular regulatory proteins such as p53, p16(INK4a), and nuclear factor kappaB (NF-kappaB). In this study, HPV DNA was detected by in situ hybridization (ISH) and p53, p16(INK4a), and NF-kappaB by immunochemistry in 22 penile cancer cases in Kenya. HPV DNA was found in 68.2% of the cases. There was no difference in the p53- and p16(INK4a)-positivities in HPV DNA-positive and HPV DNA-negative cases. In HPV DNA-positive cases, the NF-kappaB positivity in the nucleus, cytoplasm, and nucleus and/or cytoplasm amounted to 73.3%, 93.3%, and 100%, respectively, while in HPV DNA-negative cases, a 28.7% NF-kappaB positivity of in the nucleus and/or cytoplasm was observed. It is concluded that NF-kappaB in penile cancer is expressed more frequently in the presence of HPV infection than in its absence.

Autologous fat injection therapy including a high concentration of adipose-derived regenerative cells in a vocal fold paralysis model: animal pilot study.

OBJECTIVES: To verify the effectiveness and safety of the addition of adipose-derived regenerative cells to autologous fat injection therapy. METHODS: Unilateral vocal fold paralysis models were made by cutting the right recurrent laryngeal nerve in two pigs. At day 30, 0.5 ml adipose-derived regenerative cells mixed with 1 ml autologous fat was injected into the right vocal fold of one pig, with the other receiving 0.5 ml Ringer's solution mixed with 1 ml autologous fat. At day 120, fibrescopy, laser Doppler flowmeter, computed tomography, vocal function evaluation and histological assessment were conducted. RESULTS: Although histological assessment revealed atrophy of the thyroarytenoid muscle fibre in both pigs, there was remarkable hypertrophy of the thyroarytenoid muscle fibre in the area surrounding the adipose-derived regenerative cells injection site. CONCLUSION: The addition of a high concentration of adipose-derived regenerative cells to autologous fat injection therapy has the potential to improve the treatment outcome for unilateral vocal fold paralysis.

RET tyrosine kinase enhances hair growth in association with promotion of melanogenesis.

We first demonstrated that c-Ret protein is transiently expressed mainly in the inner and outer root sheaths of hair follicles soon after birth in the skin of normal C57BL/6 and BALB/c mice. A longer-lasting expression of activated RET protein overlapped the c-Ret expression with some preferential expression in the outer root sheath in close association with increase in the number of S-100 protein-containing cells in the area and excess melanogenesis in and around hair bulbs in the skin of RFP-RET-transgenic mice on a C57BL/6 background (RFP-RET/B6). Hair follicles in the skin of the transgenic mice continuously showed histology of the anagen phase, and the recovery period for the hair of the transgenic mice after shaving was shortened. Such growth promotion was not observed in the case of white hairs of RFP-RET-transgenic mice on a BALB/c background. These results suggest that RET works to extend the anagen phase in association with upregulation of melanin production.

Hypoxic enhancement of type IV collagen secretion accelerates adipose conversion of 3T3-L1 fibroblasts.

Hypoxic modulation of collagen metabolism appears to be related to pathogenesis of many diseases such as fibrosis of connective tissue after injury and scleroderma. Since most of our understanding of how procollagen assembles within the cell has come from studies on cells cultured under normoxia, it may not be helpful for the etiology of the diseases observed in peripheral tissues under hypoxic conditions. As an experimental model for the hypoxic modulation of collagen metabolism, we cultured 3T3-L1 fibroblasts under low partial oxygen pressure and found that hypoxia enhances secretion of type IV collagen 10-fold and accelerates adipose conversion of the cells. The enhanced secretion of type IV collagen was not accompanied by an appreciable increase of alpha1(IV) and alpha2(IV) mRNAs. Prolyl 4-hydroxylase alpha increased only 3-fold under hypoxia. We suggest that hypoxia creates an environment of prolyl 4-hydroxylase alpha(2)beta(2) tetramers favorable for the folding of type IV procollagen which has many interruptions of the Gly-Xaa-Yaa repeat.

Variations in the distribution, frequency, and phenotype of Langerhans cells during the evolution of malignant melanoma of the skin.

We examined the frequency, distribution, and immunophenotype (S-100 protein, CD1) of epidermal Langerhans cells (LC) in the epidermis overlying primary melanoma, and dermal dendritic cells (DC) in the dermis deep to melanoma, and in adjacent normal skin. There is a substantial reduction in S-100+ LC and a lesser decline in CD1+ LC in the epidermis over melanoma. There is a simultaneous increase in the frequency of cells expressing these phenotypes in the dermis deep to tumor. Double-staining studies in progress show coexpression of S-100 and CD1 in most of these cells. The depletion of S-100+/CD1+ DC from the peritumoral epidermis is maximum in deeper (Clark level III-V) and thicker (> 0.76 mm) tumors. The reduction in total DC in peritumoral epidermis is, however, proportionally less than the reduction in S-100+/CD1+ DC owing to an increase in cells that are S-100-/CD1+. The proportion of S-100+/CD1+ dermal DC deep to tumor is similar to that in normal epidermis and dermis, suggesting that there is no increased migration of S-100-/CD1+ DC from tumor-associated epidermis to subjacent dermis. Non-dendritic leukocytes in the dermis deep to tumor were increased in frequency, maximally in the dermis deep to thinner and more superficial melanomas. Most such cells were T lymphocytes with helper/inducer cells predominating. The data presented show substantial alterations in the frequency, distribution, and phenotype of LC/DC as melanoma evolves, alterations that may be critical for development of effective tumor-directed immunity.

Direct cloning of the Brassica S locus by using a P1-derived artificial chromosome (PAC) vector.

Self-incompatibility of Brassica is regulated by the S locus, which contains several genes including SLG and SRK. We found that both SLG and SRK genes were located at an approx. 80-kb MluI fragment in an S9 haplotype of B. campestris. Therefore, we cloned this MluI fragment into a BssHII site of the P1-derived artificial chromosome (PAC) vector. The utility of the direct cloning method is discussed in this study.

Molecular cloning of a cDNA encoding a pollen extracellular protein as a potential source of a pollen allergen in Brassica rapa.

A polyclonal antiserum was raised against the extracellular pollen proteins of Brassica rapa and used for screening the expression cDNA libraries made from immature anthers. We obtained five groups of cDNA clones, including cDNAs similar to PCP1, thioredoxin, and lipid transfer protein (LTP). Recombinant protein of the cDNA clone showing sequence similarity to LTP was demonstrated to bind IgE of a patient allergic to Brassica pollen. The cDNA clone reported here, therefore, represents a novel pollen allergen of Brassica rapa.

Engineering of hypoallergenic mutants of the Brassica pollen allergen, Bra r 1, for immunotherapy.

The Brassica pollen allergen Bra r 1 belongs to a new family of Ca2+-binding proteins, characterized by the presence of two potential EF-hand calcium-binding domains. Disruption of these EF-hand motifs by amino acid substitutions demonstrated that both domains of Bra r 1 constitute functional Ca2+-binding sites. Calcium-binding deficient mutants displayed significantly reduced IgE-binding activity. Injection of these mutated Bra r 1 variants into a murine model system showed that mouse IgG raised against the mutants recognized native Bra r 1 in Brassica pollen extracts suggesting the potential use of the engineered allergens for effective immunotherapy.

Functional transformation of plant peroxisomes.

Peroxisomes in higher plant cells are known to differentiate into at least three different classes, namely, glyoxysomes, leaf peroxisomes, and unspecialized peroxisomes, depending on the cell types. In germinating fatty seedlings, glyoxysomes that first appear in the etiolated cotyledonary cells are functionally transformed into leaf peroxisomes during greening. Subsequently, the organelles are transformed back into glyoxysomes during senescence of the cotyledons. Flexibility of function is a distinct feature of plant peroxisomes. This article briefly describes recent studies of the regulatory mechanisms involved in the changes of the function of plant peroxisomes.

Flow cytometric DNA analysis of vascular soft tissue tumors, including African endemic-type Kaposi's sarcoma.

Vascular tumors of the soft tissue display a wide spectrum of histologic features and biologic behavior. Flow cytometric DNA analysis was performed on 40 vascular tumors, including nine African endemic-type Kaposi's sarcomas, nine angiosarcomas, seven hemangiopericytomas, six glomus tumors, and nine capillary hemangiomas. Six of the nine angiosarcoma cases (67%) and one of the seven hemangiopericytomas cases (14%) were aneuploid. All benign vascular tumors and Kaposi's sarcomas were diploid. Clinically, five of the six angiosarcoma patients with aneuploidy died within 2 to 28 months, while the remaining patient, who had the smallest tumor (2 x 1 cm), survived more than 4 years after the initial diagnosis was made. All three angiosarcoma patients with diploidy died within 10 to 14 months. One hemangiopericytoma patient with aneuploidy died within 1 month. No cases of benign tumor recurred. These results suggest that most vascular tumors, which generally follow a benign clinical course, were diploid and that the majority of those with a poor outcome were aneuploid. However, flow cytometrically assessed DNA ploidy has no prognostic value in angiosarcomas or hemangiopericytomas.

Midkine, a retinoic acid-inducible heparin-binding cytokine in inflammatory responses: chemotactic activity to neutrophils and association with inflammatory synovitis.

Midkine (MK) is a retinoic acid-inducible heparin-binding cytokine. In the inflammatory synovitis of rheumatoid arthritis and osteoarthritis, MK was detected in synovial fluid, synoviocytes, and endothelial cells of new blood vessels. Normal synovial fluid and noninflammatory synovial tissue did not contain detectable MK. Therefore, MK showed inflammation-associated expression in these cases. Furthermore, MK was found to promote chemotaxis of neutrophils in the range of 10 ng/ml. The mode of action of MK was found to be haptotactic; the substrate-bound form of MK was the active one. MK is also known to promote fibrinolysis. These activities of MK are in agreement with the modes of MK expression in various pathological statuses, and thus MK is proposed to be an important molecule regulating inflammatory responses.

A comparison of epidemiologic, histologic, and virologic studies on Hodgkin's disease in western Kenya and Nagasaki, Japan.

Rapid progress in molecular technologies has enabled the detection of several oncogenic viruses in various types of tumors. The pathogenesis of Hodgkin's disease is suggested to have a strong association with Epstein-Barr virus (EBV). However, Hodgkin's disease related to EBV shows a wide geographic variation in epidemiology. These variations among different populations suggest an interaction of environmental factors and a direct role of EBV infection. Therefore, we performed a comparative study on epidemiologic, histologic, and virologic features of Hodgkin's disease among those in the western part of Kenya and in Nagasaki, Japan. The age distribution of Hodgkin's disease showed a distinct peak in the 0-9-year-old age group in Kenya, and a higher and lower peak in the 60-69- and 30-39-year-old age groups, respectively, in Japan. The most common subtype of Hodgkin's disease in both countries was mixed cellularity, followed by nodular sclerosis, lymphocyte depletion, and lymphocyte predominance. Mixed cellularity showed a significantly high prevalence among Kenyan children nine years of age or younger. Using the in situ hybridization method, EBV-encoded RNA (EBER-1) was detected in 79% of the Kenyan cases and 59% of the Japanese cases, with the mixed cellularity subtype showing a strong correlation with EBER-1. There was 100% positivity in both countries in those less than nine years old. These results suggest that EBV plays a more direct role in the pathogenesis of Hodgkin's diseases in Kenya, especially in cases among young children and also in Japanese children. Environmental and/or genetic factors may have a role, in addition to EBV, in the pathogenesis of Hodgkin's disease, especially in Nagasaki, Japan.

The pattern of pediatric solid malignant tumors in western Kenya, east Africa, 1979-1994: an analysis based on histopathologic study.

This study analyzed histopathologic specimens of 600 pediatric solid malignant tumors seen during the period 1979-1994 at the histopathology laboratories of the Rift Valley Provincial General Hospital in Nakuru, the Nyanza Provincial General Hospital in Kisumu, and the Uasin Gishu Hospital in Eldoret in western Kenya. The crude incidence rate of each malignancy per 100,000 children per year was calculated. The patterns of malignancies were examined with a focus on tumor incidence, age, sex, geographic, and ethnic distribution to relate the tumors to putative environmental and genetic causative factors. The six common tumors were Burkitt's lymphoma (33.5%), non-Hodgkin's lymphoma (21.8%), retinoblastoma (11.5%), Kaposi's sarcoma (6.1%), nephroblastoma (4.5%), and Hodgkin's disease (4.1%). Significantly high crude incidence rates for lymphomas and Kaposi's sarcoma showed a characteristic ethnogeographic distribution. The majority of the tumors were found concentrated around Lake Victoria and showed decreasing occurrence as one moved towards the semi-arid and highland areas. We concluded that environmental factors seem to play a major role in childhood tumors in western Kenya.

Short report: herpes-like DNA sequences in African-endemic and acquired immunodeficiency syndrome-associated Kaposi's sarcoma.

Recently, the unique nucleic acid closely related to the herpes-like sequences has been found in acquired immunodeficiency syndrome (AIDS)-associated Kaposi's sarcoma (KS). We have confirmed the presence of herpes-like DNA sequences in six cases of AIDS-associated KS and three of the nine cases of African-endemic KS in adults, but not in eight cases of KS in children from the same area. These sequences were seen in a histologically early stage of KS. Our results suggest that herpes-like DNA sequences may play an important role in the pathogenesis of AIDS-associated KS.

The existence of distinct classes of prostaglandin E2 receptors mediating adenylate cyclase and phospholipase C pathways in osteoblastic clone MC3T3-E1.

We have reported previously that PGE2 evoked an increase in intracellular calcium level ([Ca2+]i) in mouse osteoblastic cells (1). Here, we investigated the effects of PGE1 and PGF2 alpha on cAMP production and [Ca2+]i in comparison with those of PGE2. In osteoblastic clone, MC3T3-E1 cells, PGE1 stimulated cAMP production, but had no effect on [Ca2+]i, whereas PGF2 alpha evoked only [Ca2+]i increase. In contrast, PGE2 not only stimulated cAMP production, but also increased [Ca2+]i. From the Scatchard plot analysis of PGE2 it was confirmed that there were two classes of PGE2 binding sites (Kd value, 9.2 nM; binding site, 29 fmole/mg protein, and Kd value, 134 nM; binding site, 148 fmole/mg protein). As the increase in [Ca2+]i was caused by PGF2 alpha and PGE2, but not by PGE1, we investigated the displacement of [3H]-PGF2 alpha binding. The displacement capacity of unlabeled PGE2 was about 110 of that of PGF2 alpha, while that of PGE1 was very low even at 500-fold excess. These data indicate the possibility that the dual action of PGE2 is mediated by distinct receptor systems.

Difference in histological activity of non-tumorous liver tissue between hepatitis B virus- and hepatitis C virus-associated hepatocellular carcinoma without cirrhosis.

To elucidate the difference in the liver carcinogenetic process during hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, non-tumorous liver tissues obtained from 10 patients who developed HBV-associated hepatocellular carcinoma (HCC) without cirrhosis were compared with those obtained from 26 patients who developed HCV-associated HCC without cirrhosis. The extent of fibrosis was similar in both groups. In contrast, necroinflammatory activities were significantly higher in patients with HCV than in patients with HBV. These results indicate that ongoing liver inflammation mediates the hepatocarcinogenesis more pronouncedly in HCV infection than in HBV infection.

Tapetum-specific expression of theOsg6B promoter-ß-glucuronidase gene in transgenic rice.

The promoter of an anther tapetum-specific gene,Osg6B, was fused to aß-glucuronidase (GUS) gene and introduced into rice byAgrobacterium-mediated gene transfer. Fluorometric and histochemical GUS assay showed that GUS was expressed exclusively within the tapetum of anthers from the uninucleate microspore stage (7 days before anthesis) to the tricellular pollen stage (3 days before anthesis). This is the first demonstration of an anther-specific promoter directing tapetum-specific expression in rice.

Absorption, distribution and excretion of neocarzinostatin (NCS) in mice after oral administration.

Distribution, excretion and toxicity of an antitumor protein, neocarzinostatin (NCS) were examined in mice after oral administration. The oral LD50 1 g/kg after intravenous injection. After oral administration of 200 mg/kg of NCS, the tissue level was low but detectable in lung, skin and pancreas in addition to the tissues of the gastrointestinal tract. The NCS level in lung and skin remained constant through 6 hours. In gastrointestinal tissues after oral administration the level was higher in the stomach than the large intestine or small intestine. The total recovery of orally administered NCS in feces of mice was 26.5% of the given dose during the first 12 hours. Inactivation of NCS by homogenates of small and large intestines (about 50%) was found in in vitro experiments.

Expression of midkine in normal and burn sites of rat skin.

Expression of midkine (MK), a retinoic acid-inducible heparin binding growth factor, was examined immunohistochemically in normal and burn sites of rat skin. In the normal skin, MK was localized in the epidermis and dermal appendages such as hair follicles and sebaceous glands. Mast cells in the subdermal connective tissue also accumulated MK. After burn injury, MK-positive cells began to infiltrate into subdermal connective tissue, and the number of MK-positive cells in the region increased to a maximum at postburn day two and then decreased gradually. Western blotting analysis of both normal and postburn skin revealed a 30 kDa band reactive with anti-MK antibody; this band was concluded to be a dimer of MK. These findings were discussed from the viewpoint of the possible role of MK in wound healing.