Polymyxin-B hemoperfusion for acute exacerbation of idiopathic pulmonary fibrosis: serum IL-7 as a prognostic marker.

BACKGROUND: Acute exacerbation (AE) of idiopathic pulmonary fibrosis (IPF) has an extremely poor prognosis. Direct hemoperfusion with a polymyxin B-immobilized fiber column (PMX-DHP) has been used to improve oxygenation for acute respiratory distress syndrome. The study aim was to retrospectively determine the predictive factors affecting the prognosis of AE of IPF treated with PMX-DHP. METHODS: We studied patients suffering from AE of IPF, treated with PMX-DHP combined with high-dose corticosteroid therapy. Stored serum taken before and after PMX-DHP therapy was analyzed for 27 cytokines and chemokines. RESULTS: Nineteen patients with AE of IPF were studied. The median survival time after diagnosis of AE was 22 days. Survival rates after diagnosis of AE were 47.4% at 30 days, 31.6% at 60 days, and 26.3% at 90 days. Serum levels of Interleukin (IL)-7, an anti-fibrotic cytokine, in survivors at day 30 following PMX-DHP therapy ('Survivors') significantly increased after the treatment, compared to serum levels of non-survivors at day 30 after the therapy ('Nonsurvivors'), which did not demonstrate a significant change. Serum levels of IL-1beta, interferon-y and chemokine ligand (CCL) 2 levels were not significantly altered in 'Survivors', but were significantly changed in 'Nonsurvivors.' Multivariate Cox proportional-hazards analysis showed that an increase in IL-7 levels after PMX-DHP therapy and treatment without intubation (other than invasive positive-pressure ventilation) were significantly better prognostic factors. CONCLUSION: The results suggest that serum IL-7 may be a useful prognostic factor for patients with AE of IPF treated with PMX-DHP, possibly reflecting underlying anti-fibrotic mechanisms.

Sample collection from asteroid (162173) Ryugu by Hayabusa2: Implications for surface evolution.

The near-Earth asteroid (162173) Ryugu is thought to be a primitive carbonaceous object that contains hydrated minerals and organic molecules. We report sample collection from Ryugu's surface by the Hayabusa2 spacecraft on 21 February 2019. Touchdown images and global observations of surface colors are used to investigate the stratigraphy of the surface around the sample location and across Ryugu. Latitudinal color variations suggest the reddening of exposed surface material by solar heating and/or space weathering. Immediately after touchdown, Hayabusa2's thrusters disturbed dark, fine grains that originate from the redder materials. The stratigraphic relationship between identified craters and the redder material indicates that surface reddening occurred over a short period of time. We suggest that Ryugu previously experienced an orbital excursion near the Sun.

Characterization of a leucine aminopeptidase of Babesia gibsoni.

Peptidases of parasitic protozoa are currently under intense investigation in order to identify novel virulence factors, drug targets, and vaccine candidates, except in Babesia. Leucine aminopeptidases in protozoa, such as Plasmodium and Leishmania, have been identified to be involved in free amino acid regulation. We report here the molecular and enzymatic characterization, as well as the localization of a leucine aminopeptidase, a member of the M17 cytosolic aminopeptidase family, from B. gibsoni (BgLAP). A functional recombinant BgLAP (rBgLAP) expressed in Escherichia coli efficiently hydrolysed synthetic substrates for aminopeptidase, a leucine substrate. Enzyme activity of the rBgLAP was found to be optimum at pH 8.0 and at 37 degrees C. The substrate profile was slightly different from its homologue in P. falciprum. The activity was also strongly dependent on metal divalent cations, and was inhibited by bestatin, which is a specific inhibitor for metalloprotease. These results indicated that BgLAP played an important role in free amino acid regulation.

Prevalence of Theileria equi and Babesia caballi infections in horses belonging to resource-poor farmers in the north-eastern Free State Province, South Africa.

The prevalence of Theileria equi and Babesia caballi infections in the north-eastern Free State Province of South Africa was determined by examination of thin and thick Giemsa-stained blood smears, IFAT and PCR. No parasites were detected by microscopy from any blood samples collected at five study sites, Qwaqwa, Kestell, Harrismith, Vrede and Warden. Of the tested serum samples, 28/29 (96.5%), 20/21 (95.2%) and 42/42 (100%) were positive by IFAT for T. equi infections in Harrismith, Kestell and Qwaqwa, respectively, and 5/29 (17.2%), 13/21 (61.9%) and 30/42 (71.4%) were sero-positive for B. caballi infections in Harrismith, Kestell and Qwaqwa, respectively. All DNA samples from the study sites were negative for B. caballi infections by PCR, but five samples, two from each of Kestell and Warden and one from Vrede, were PCR positive for T. equi infections. The high prevalence of antibodies against T. equi and B. caballi in the sampled horses indicates that the animals had been exposed to T. equi and B. caballi infections but the absence of parasitaemia and very low number of positive PCR samples, however, imply that T. equi and B. caballi are endemically stable in the north-eastern Free State Province.

Preliminary application and evaluation of loop-mediated isothermal amplification (LAMP) for detection of bovine theileriosis and trypanosomosis in Tanzania.

The sensitivity of LAMP, PCR and microscopy to detect Theileria spp. and Trypanosoma congolense in field-derived bovine blood samples from Tanzania was evaluated and compared. No parasites were detected by microscopy. Furthermore, no bovine Theileria spp. were detected by LAMP and PCR from all the 24 samples collected from Arusha. Four and one out of 24 samples were positive for Theileria congolense infection by LAMP and PCR respectively while, 18 and nine out of 40 samples from Dar es Salaam were positive by LAMP and PCR for Theileria spp. Infection, respectively. Although all samples from Dar es Salaam were negative for Trypanosoma congolense infections by PCR, 12 out of 40 samples were LAMP positive. Whilst PCR is an established gene amplification method for the detection of Theileria and trypanosome parasites, this study introduces LAMP as an alternative molecular diagnostic tool that could be used in large-scale epidemiological surveys.

Prevalence and risk factors of bovine trypanosomosis in Kilwa district, Lindi region of southern Tanzania.

African animal trypanosomosis (AAT) and human African trypanosomosis (HAT) are complex chronic, debilitating, emaciating and often fatal diseases of animals and humans, respectively. This cross-sectional study was conducted to determine the prevalence and risk factors associated with bovine trypanosomosis in tsetse-infested Kilwa district, Lindi region, southern Tanzania. Blood samples were collected from 420 cattle randomly selected from 86 herds from ten villages. A maximum of ten herds per village and at most six animals from each herd were selected for sampling. At the same time, a questionnaire was administered. Individual animal samples were analysed using microscopy and pooled sample at herd level were analysed by loop mediated isothermal amplification (LAMP). A herd was considered positive if at least one animal in the herd was positive for AAT. A prevalence of 9.3% (95% CI: 2.9-14.9) was recorded for AAT by microscopy, mainly caused by Trypanosoma congolense (5.8%, 95% CI=0.9-10.7), Trypanosoma brucei species (5.8%, 95%, CI=0.9-10.7) and Trypanosoma vivax (3.5%, 95% CI=0-7.4). Loop mediated isothermal amplification (LAMP) recorded a heard prevalence of 41.9% (95% CI: 30.0-51.4%), mainly caused by T. congolense (30.2%, 95% CI: 20.5-39.9), T. brucei species (25.6%, 95% CI: 16.4-34.8) and T. vivax (20.9%, 95% CI: 12.3-29.7). Most of the cattle herds had mixed infections of these parasites. According to LAMP, Miteja and Matandu villages had the highest AAT herd prevalence of 57% (95% CI: 20.3-93.7) while Mavuji had the lowest prevalence of 14% (95% CI: 0-39.7). Data from the present study suggest that district of origin, grazing in game reserve, water source and form of watering point are risk factors associated with AAT in Kilwa district, southern Tanzania. Continuous surveillance and monitoring of AAT using more sensitive are recommended.

Characterization of an 84 kDa protein inducing an immediate hypersensitivity reaction in rabbits sensitized to Haemaphysalis longicornis ticks.

An immunogenic 84 kDa protein was isolated and purified from whole tick extracts of Haemaphysalis longicornis larvae by a combination of ion exchange, reverse phase and hydrophobic interaction chromatographies. The protein, when injected intradermally into rabbits exposed to repeated tick feeding, induces an immediate cutaneous hypersensitivity reaction. It has been purified to homogeneity as shown by sodium dodecyl sulphate polyacrylamide gel electrophoresis and silver staining. Amino acid sequences for two peptides derived from proteolytic cleavage of p84 were scanned against known proteins on the SWISS-PROT database. A 7 residue motif, ISGWGNT present in one of the two peptides appeared conserved in both vertebrate and invertebrate trypsin-like serine proteinases, while another 7 amino acid motif, HVPAGQI present in the second peptide showed homology to an Escherichia coli ATP-binding protein. We have discussed our findings in relation to isolation and characterization of target antigens for tick vaccine candidates.

Apoptosis in peripheral CD4+T cells and thymocytes by Marek's disease virus-infection.

Histological study revealed that Marek's disease virus (MDV) can cause apoptosis in peripheral blood lymphocytes (PBL) in latently infected chickens. Analysis of DNA fragmentation indicated that CD4+T cells but not CD8+T cells underwent apoptosis. These apoptotic changes were also observed in the thymus during the acute phase of the infection. Flow cytometry analysis showed the drastic decrease of CD4+CD8+ thymocytes, indicating that MDV can induce apoptosis in CD4+CD8+ immature thymocytes in acutely infected chickens. These changes might be involved in the immuno-suppression induced by MDV.

Prevalence and species distribution of caprine trypanosomosis in Sinazongwe and Kalomo districts of Zambia.

African animal trypanosomosis is one of the key livestock diseases hindering full exploitation of livestock production potential covering 37 countries across sub-Saharan Africa. Many studies have been carried out to investigate the prevalence of the disease in cattle and humans in many tropical African countries but very little attention has been directed towards generating the disease prevalence rates in goats. The current study was conducted between December 2013 and January 2014 to establish the prevalence of caprine trypanosomosis in Sinazongwe and Kalomo districts, southern Zambia. It involved 422 goats which were first examined by palpation for possible enlargement of superficial lymph nodes. Blood samples were then collected from the goats and subjected to laboratory diagnosis using the microscope and Loop Mediated Isothermal Amplification (LAMP). None of the examined goats displayed enlargement of superficial lymph nodes. On microscopy only one goat was found to be positive. The results of investigation using the LAMP method showed that 100 goats were infected with trypanosomes giving an overall prevalence rate of 23.7%. The prevalence of infection in Sinazongwe was 22.4% (n=183) while in Kalomo it was 24.7% (n=239); and the difference between the two districts was statistically significant at 95% CL (x(2)=4.4, df=1, p<0.05). Trypanosoma brucei, Trypanasoma vivax and Trypanasoma congolense were detected in 82.0%, 31.0% and 23.0% of the infected goats, respectively. Mixed infections were detected among 33.0% of the positive samples. The high prevalence rate of trypanosomes detected in the study area confirms the earlier reports that trypanosomosis is re-emerging in the areas previously aerial sprayed by Government. The detection of trypanosomes in naturally infected goats outlines the important role goats play in the epidemiology of African animal trypanosomosis.

Molecular screening for Rickettsia, Anaplasmataceae and Coxiella burnetii in Rhipicephalus sanguineus ticks from Malaysia.

A total of 44 Rhipicephalus sanguineus ticks collected from 23 dogs from Malaysia were screened for Rickettsia, Anaplasmataceae and Coxiella burnetii. Coxiella burnetii was detected in 59% (26/44) of ticks however Rickettsia and Anaplasmataceae were not detected in any of the ticks. In order to genotype the strains of C. burnetii, multispacer sequence typing (MST) was carried out using three different spacers. One of the spacers; Cox2 successfully amplified a fragment for which the full length sequence of 397 bp was obtained. The sequenced product revealed only a single nucleotide difference with the Cox2.3 type sequence.

Issues in tick vaccine development: identification and characterization of potential candidate vaccine antigens.

It is well established that acquired immunity against tick infestation can be induced by repeated tick infestation or by active immunization with either crude or purified native as well as recombinant antigens. This review provides insights into the development of tick vaccines with reference to identification, purification and molecular cloning of candidate target antigens.

Expression of two subtypes of human IFN-alpha in transgenic potato plants.

Plant expression systems have advantages over other in vitro expression systems in terms of low production costs and low risk of contamination by animal viruses or bacterial endotoxins. In this study, cDNA encoding two subtypes of human interferon-alpha2b and 8 (HuIFN-alpha2b and HuIFN-alpha8) were introduced into potato plants (Solanum tuberosum) using Agrobacterium-mediated transformation. Transcription and translation of the inserted HuIFN-alpha cDNA were confirmed by Northern blot analysis and ELISA, respectively. Bioactivity of the products was assayed by inhibition of vesicular stomatitis virus (VSV) replication on a human amniotic cell line. However, because of the presence of substances in potato tissue extracts that were toxic to animal cells, successful demonstration of IFN bioactivity in the transformants was achieved only after removal of such substances by dialysis. The maximum level of IFN activity in plant extracts was 560 IU/g of tissue. These results indicated that the HuIFN-alpha gene introduced into the potato plant was correctly translated and transcribed in plant cells. This report for the first time shows that biologically active animal cytokines with potential pharmaceutical applications could be expressed in transgenic potato plants.

Analysis of the genes encoding immunodominant piroplasm surface proteins of Theileria sergenti and Theileria buffeli by nucleotide sequencing and polymerase chain reaction.

The nucleotide sequences of the cDNAs encoding a 33-kDa piroplasm protein of Theileria sergenti (p33) and a similar protein of Theileria buffeli (p34) were determined. Both of the genes contained an open reading frame of 849 base pairs. The predicted amino acid sequence of p33 and p34, consisting of 283 residues, showed 82% similarity. A transmembrane hydrophobic domain and signal peptides were predicted. The polymerase chain reaction was used to amplify p33/34 genes from the piroplasm DNA of T. sergenti, T. buffeli and Theileria orientalis. Following amplification, p33 and p34 genes were clearly differentiated using the restriction enzymes sites that were not shared between them. These results indicated that p33 and p34 were conserved molecules among these Theileria species, and the genes that encode p33/34 proteins were suitable for discrimination of T. sergenti from T. buffeli/T. orientalis.

Protein changes in bovine lymphoblastoid cells induced by infection with the intracellular parasite Theileria parva.

Protein and glycoprotein changes induced in bovine lymphoblasts by infection with Theileria parva were analyzed by high-resolution two-dimensional gel electrophoresis. Uninfected and infected cloned bovine T and B lymphoblasts were biosynthetically labeled with [35S]methionine and their two-dimensional autoradiographic patterns were compared with each other and with the pattern obtained using purified labeled schizonts. Ten proteins were found in infected cells which were not present in uninfected cells, and seven of these were detected in preparations of purified schizonts. Four glycoproteins were detected on the surface of infected cells labeled with [3H]borohydride while a major glycoprotein present on uninfected cells disappeared or was reduced in infected cells. Other minor changes in protein and glycoprotein patterns were also observed.

Conventional tumor markers are prognostic indicators in patients with head and neck squamous cell carcinoma.

We tested for squamous cell carcinoma-related antigen (SCC), carcinoembryonic antigen (CEA), ferritin, immunosuppressive acid protein (IAP) and sialic acid in the serum from 247 patients with head and neck squamous cell carcinoma prior to therapy. Significant correlations were found between IAP and tumor size, lymph node metastasis, and clinical stage (P<0.0001, P<0.001, and P<0.0001). Also, sialic acid and SCC were also correlated with tumor size, lymph node metastasis, and clinical stage. Moreover IAP, sialic acid and SCC were strongly associated with survival rate (P<0.0001, P = 0.0230 and P = 0.0159). A multivariate Cox proportional hazard model demonstrated that being positive for IAP was an independent predictor for patients with H&NSCC (P = 0.0115). The results indicate that IAP, sialic acid and SCC are useful as prognostic factors.

Expression of hMSH2 correlates with in vitro chemosensitivity to CDDP cytotoxicity in oral and oropharyngeal carcinoma.

We investigated the expression of hMSH2, a human mutS homologue from chromosome 2p, in oral and oropharyngeal squamous cell carcinoma (SCC) by an immunohistochemical technique and performed tumor in vitro chemosensitivity testing. In 58 oral and oropharyngeal SCC, the hMSH2 positive score was inversely associated with tumor size, but not with other clinical parameters. Among five anticancer drugs (cisplatin (CDDP), 5-FU, peplomycin, mitomycin C and doxorubicin), only for CDDP was sensitivity to cytotoxicity correlated with the hMSH2 positive score. The susceptibility of hMSH2-positive tumors to CDDP killing was significantly higher than that of hMSH2-negative tumors. Immunohistochemical results regarding hMSH2 are promising in the evaluation of the sensitivity of cancer cells to CDDP cytotoxicity and enable one to select patients for adjuvant chemotherapy for oral and oropharyngeal SCC.

Decreased expression of Bax is correlated with poor prognosis in oral and oropharyngeal carcinoma.

We investigated the expression of apoptosis-related factors, p53, Bax, Bcl-2, and spontaneous apoptosis in 57 cases of oral and oropharyngeal squamous cell carcinoma (SCC) by immunochemical staining and ApopTag kit. Positive expression of Bax was inversely associated with advanced tumor stage (P = 0.0225), lymph node metastasis (P = 0.0225), clinical stage (P = 0.0083) and poor prognosis (P = 0.0478). Positive expression of p53 was related to poor prognosis (P = 0.0445) and was associated with negative expression of Bax (P = 0.0439). The apoptosis index did not correlate with clinical outcome. These results suggest that abnormality of Bax expression plays an important role in tumor progression in oral and oropharyngeal SCC.

Expression of multidrug resistance-associated protein (MRP) in head and neck squamous cell carcinoma.

We investigated the expression of multidrug resistance-associated protein (MRP) in 115 cases of head and neck squamous cell carcinoma (H&NSCC) by immunohistochemistry and examined the relationship between MRP expression and clinical factors. Thirty-four (30%) of 115 cases of H&NSCC had expression of MRP. The clinical stage was inversely associated with the expression of MRP (P = 0.0090), but not with age, sex, tumor size, metastasis, recurrence, death from disease or overall survival rate for 5 years. In vitro chemosensitivity to five chemotherapeutic agents (cis-diamminedichloroplatinum, 5-fluorouracil, peplomycin, mitomycin C and Adriamycin) was tested by ATP assay and no correlation between the sensitivity of tumor cells to the cytotoxicity of any drug and MRP expression was found. These results suggest that the resistance to anticancer drugs is not dependent only on the expression of MRP in H&NSCC.

Enhancement of cisplatin sensitivity and platinum uptake by 40 degrees C hyperthermia in resistant cells.

We studied the cytotoxic and pharmacological properties of 40 degrees C hyperthermia and CDDP in CDDP-sensitive (IMC-3) and CDDP-resistant (IMC-3-DDP) human maxillary carcinoma cells. Heating at 40 degrees C alone caused almost no cell killing to IMC-3 and IMC-3-DDP cells. In both cell lines, the dose-dependent cytotoxicity of 2-h exposures to CDDP was increased at 40 degrees C in comparison to 37 degrees C. Heating at 40 degrees C also potentiated CDDP cytotoxicity in both IMC-3 and IMC-3-DDP cells with thermal chemoenhancement ratios (CER) of 1.48 and 1.94, respectively. The intracellular CDDP uptake level of IMC-3-DDP at 37 degrees C was significantly reduced compared with IMC-3 cells. At 40 degrees C, however, hyperthermia increased platinum accumulation by factors of 1.4 and 1.8 in IMC-3 and IMC-3-DDP cells, respectively. These findings indicated that CDDP sensitivity was hyperthermically chemopotentiated in CDDP-resistant variants rather than in the control clones. Thus, clinical cancer chemotherapy with CDDP may be improved by an appropriate combination with hyperthermia even at 40 degrees C.

Expression of fas (CD95) ligand is correlated with IL-10 and granulocyte colony-stimulating factor expression in oral and oropharyngeal squamous cell carcinoma.

The Fas/Fas ligand (FasL) pathway has been shown to be an important cellular pathway mediating apoptosis. In this study we investigated the expression of Fas and FasL and the rate of spontaneous apoptosis in 58 oral and oropharyngeal squamous cell carcinoma (SCC) by using immunohistochemical techniques. There was no correlation between Fas or FasL expression and clinicopathological factors. The expression of Fas in the tumor did not affect spontaneous apoptosis of the tumor cells. However, FasL expression was associated with IL-10 and granulocyte colony-stimulating factor expression in oral and oropharyngeal SCC. These results suggested that the Fas/FasL system is connected with the expression of various factors including cytokines in tumor cells.