Th1 immune response to Plasmodium falciparum recombinant thrombospondin-related adhesive protein (TRAP) antigen is enhanced by TLR3-specific adjuvant, poly(I:C) in BALB/c mice.

Sporozoite-based malaria vaccines have provided a gold standard for malaria vaccine development, and thrombospondin-related adhesive protein (TRAP) serves as the main vaccine candidate antigen on sporozoites. As recombinant malaria vaccine candidate antigens are poorly immunogenic, additional appropriate immunostimulants, such as an efficient adjuvant, are highly essential to modulate Th1-cell predominance and also to induce a protective and long-lived immune response. In this study, polyinosinic:polycytidylic acid [poly(I:C)], the ligand of TLR3, was considered as the potential adjuvant for vaccines targeting stronger Th1-based immune responses. For this purpose, BALB/c mice were immunized with rPfTRAP delivered in putative poly(I:C) adjuvant, and humoural and cellular immune responses were determined in different immunized mouse groups. Delivery of rPfTRAP with poly(I:C) induced high levels and titres of persisted and also high-avidity anti-rPfTRAP IgG antibodies comparable to complete Freund's adjuvant (CFA)/incomplete Freund's adjuvant (IFA) adjuvant after the second boost. In addition, rPfTRAP formulated with poly(I:C) elicited a higher ratio of IFN-γ/IL-5, IgG2a/IgG1, and IgG2b/IgG1 than with CFA/IFA, indicating that poly(I:C) supports the induction of a stronger Th1-based immune response. This is a first time study which reveals the potential of rPfTRAP delivery in poly(I:C) to increase the level, avidity and durability of both anti-PfTRAP cytophilic antibodies and Th1 cytokines.

Morphological and Molecular Characteristic of Megaselia scalaris (Diptera: Phoridae) Larvae as the Cause of Urinary Myiasis.

We report a case of urinary myiasis occurring in a 60-yr-old Iranian male patient with urinary tract problems and a history of travel to Thailand who was referred to Shafagh Medical Laboratory in Tehran (Iran). Larvae excreted in the patient's urine were confirmed by morphological identification key and DNA barcoding to belong to the species Megaselia scalaris Loew, which is known as the scuttle fly. Based on the patient's history, he was infected with M. scalaris in Thailand. To the best of our knowledge, this is the first report of urinary myiasis caused by M. scalaris in Thailand.

Metastatic spread of cutaneous melanoma to the sigmoid colon: a rare but important consideration.

BACKGROUND: Cutaneous melanoma is known for its aggressive tendency for metastasis, most commonly to lymph nodes, lung, liver, and brain. CASE REPORT: We present the case of an 80-year-old male, with a history of cutaneous melanoma, found to have biopsy-proven metastatic melanoma deposits in the sigmoid colon. This rare case was initially thought to be a walled-off perforation secondary to diverticulitis. Although, computed tomography, showed features more strongly suggestive of malignancy. We propose early consideration of bowel metastasis in patients with a history of cutaneous melanoma presenting with lower gastrointestinal symptoms. CONCLUSION: Once malignancy is included in the differential for any inflammatory type bowel lesion, arranging endoscopy and biopsy is essential to confirm the diagnosis and guide management. Multidisciplinary team discussion is recommended to determine the most appropriate treatment strategy - radical versus palliative, which must always consider the patient's performance status alongside cancer staging. HIPPOKRATIA 2017, 21(4): 194-196.

The influence of obesity and body mass index on the outcome of laparoscopic colorectal surgery: a systematic literature review.

AIM: The relationship between obesity, body mass index (BMI) and laparoscopic colorectal resection is unclear. Our object was to assess systematically the available evidence to establish the influence of obesity and BMI on the outcome of laparoscopic colorectal resection. METHOD: A search of PubMed/Medline databases was performed in May 2015 to identify all studies investigating the impact of BMI and obesity on elective laparoscopic colorectal resection performed for benign or malignant bowel disease. Clinical end-points examined included operation time, conversion rate to open surgery, postoperative complications including anastomotic leakage, length of hospital stay, readmission rate, reoperation rate and mortality. For patients who underwent an operation for cancer, the harvested number of lymph nodes and long-term oncological data were also examined. RESULTS: Forty-five studies were analysed, the majority of which were level IV with only four level III (Oxford Centre for Evidence-based Medicine 2011) case-controlled studies. Thirty comparative studies containing 23 649 patients including 17 895 non-obese and 5754 obese showed no significant differences between the two groups with respect to intra-operative blood loss, overall postoperative morbidity, anastomotic leakage, reoperation rate, mortality and the number of retrieved lymph nodes in patients operated on for malignancy. Most studies, including 15 non-comparative studies, reported a longer operation time in patients who underwent a laparoscopic procedure with the BMI being an independent predictor in multivariate analyses for the operation time. CONCLUSION: Laparoscopic colorectal resection is safe and technically and oncologically feasible in obese patients. These results, however, may vary outside of high volume centres of expertise.

Administration of naloxone in combination with recombinant Plasmodium vivax AMA-1 in BALB/c mice induces mixed Th1/Th2 immune responses.

Naloxone (NLX) has the ability to shift the immune response to a Th1 profile. Therefore, the adjuvant efficacy of NLX with recombinant P. vivax apical membrane antigen-1(rPvAMA-1) in BALB/c mice was evaluated. Mice were immunized subcutaneously with purified rPvAMA-1 formulated with NLX (doses of 5 mg/kg body weight) alone or in combination with IFA. A significant increase in anti-PvAMA-1 IgG antibody after the second boost (mean OD490  = 2·08 and 2·17, in groups received, rPvAMA-1/NLX and rPvAMA-1/NLX/IFA, respectively) was detected. IgG1 and IgG2b were the predominant isotypes in all immunized mouse groups. In immunized mice with rPvAMA-1/NLX (mean: 1036 pg/mL) and with rPvAMA-1/NLX/IFA (mean: 1024 pg/mL), IFN-γ was elicited in response to rPvAMA-1 after the second boost. No detectable IL-4 secretion was determined in all tested groups. In conclusion, the administration of NLX alone or NLX/IFA with rPvAMA-1 in BALB/c mice, which induced mixed Th1/Th2 immune responses, was comparable with that of the same recombinant antigen with CFA/IFA adjuvant. The results indicate that NLX alone may possibly not be considered as a potent Th1 adjuvant in PvAMA-1-based vaccine. However, in order to modulate immune responses from mixed Th1/Th2 to strong and protective Th1 response, further study is warranted on combination of NLX with other adjuvants such as CpG motifs or MPL in proper vaccine formulation. Additionally, dose-response study is necessary to determine the effect of different doses of antigen combined with NLX (at various doses) in Balb/c mice.

Photocatalytic removal of 2-nitrophenol using silver and sulfur co-doped TiO2under natural solar light.

To overcome the drawback of poor solar light utilization brought about by the narrow photoresponse range of TiO2, a silver and sulfur co-doped TiO2was synthesized. Using the prepared catalyst, solar photocatalytic degradation of 2-nitrophenol (2-NP) by a TiO2-based catalyst was studied for the first time. Effects of the co-doping on the structural, optical and morphological properties of the synthesized nanoparticles were investigated by different characterization methods: X-ray diffraction, N2 adsorption-desorption measurements, scanning electron microscopy, transmission electron microscopy, energy-dispersive X-ray spectroscopy, UV-visible diffuse reflectance spectroscopy and Fourier transform infrared spectroscopy. Solar experiments showed that the co-doping with silver and sulfur significantly increased the photocatalytic activity. In various initial concentrations of 2-NP more than 99% of the contaminant was decomposed by Ag-S/TiO2in less than 150 minutes, while the degradation efficiency was much less in the presence of bare TiO2. Kinetic studies suggested that solar photocatalytic degradation of 2-NP is consistent with the Langmuir-Hinshelwood model. The rate constant of the reaction and adsorption constant of the modified photocatalyst were found to be 2.4 and 4.1 times larger than that of bare TiO2, respectively.

A systematic review of the literature on the surgical management of recurrent rectal prolapse.

AIM: There are no available guidelines to support surgical decision-making in recurrent rectal prolapse. This systematic review evaluated the results of abdominal or perineal surgery for recurrent rectal prolapse, with the aim of developing an evidence-based treatment algorithm. METHOD: PubMed and MEDLINE databases were searched for all clinical studies involving patients who underwent surgery for recurrent rectal prolapse between 1950 and 2014. The primary outcome measure was the recurrence rate after abdominal or perineal surgery for recurrent rectal prolapse. Secondary outcomes included morbidity, mortality and quality of life data where available. RESULTS: There were no randomized controlled studies comparing the success rates of abdominal or perineal surgery for recurrent rectal prolapse. Most studies were heterogeneous, of low quality (level IV) and involved small numbers of patients. The follow-up of 144 patients included in the studies undergoing perineal surgery ranged from 8.8 to 81 months, with recurrence rates varying from 0% to 50%. Morbidity ranged from 0% to 17% with no mortality reported. Limited data on quality of life following the Altemeier procedure were available. The follow-up for 158 patients included in the studies who underwent abdominal surgery ranged from 0 to 23 years, during which recurrence rates varied from 0% to 15%. Morbidity rates ranged from 0% to 32% with 4% mortality. No quality of life data were available for patients undergoing abdominal surgery. CONCLUSION: This systematic review was unable to develop a treatment algorithm for recurrent rectal prolapse due to the variety of surgical techniques described and the low level of evidence within heterogeneous studies. Larger high-quality studies are necessary to guide practice in this difficult area.

Gracilis muscle interposition for rectovaginal and anovaginal fistula repair: a systematic literature review.

BACKGROUND: Repair of rectovaginal fistula (RVF) is associated with high recurrence. For this reason gracilis muscle interposition is increasingly being used. AIM: To evaluate the efficacy of this procedure for RVF repair. METHOD/SEARCH STRATEGY: A search of PubMed and Medline databases was performed in November 2013 using the text terms and MESH headings 'rectovaginal fistula/fistulation', and 'gracilis muscle', spanning 1980-2013. The search strategy was restricted to articles written in English with available abstracts. Sample size, aetiology of RVF, previous repair attempts, follow-up period, healing rates and complications were recorded and analysed. RESULTS: Seventeen studies involving 106 patients were analysed. The cause of RVF included inflammatory bowel disease (n = 37 [34.9%]: Crohn's disease [34], ulcerative colitis [3]), pelvic surgery (37 [34.9%]), obstetric injury (9 [8.5%]), malignancy (7 [6.6%]), trauma (5 [4.7%]), miscellaneous (idiopathic, endometriosis, radiation: 11 [10.4%]). Patients had undergone a median number of two previous unsuccessful repairs. At a median follow-up of 21 months, healing had occurred in 33-100% (median 100%) with the largest studies reporting rates between 60% and 90%. Thirteen studies did not report any complications, with the remainder reporting only minor morbidity. CONCLUSION: Gracilis interposition appears to have a reasonable success rate for RVF repair with acceptable morbidity. It may be considered as one of the first-line treatment options for recurrent RVF.

Absence of asymptomatic malaria infection in endemic area of bashagard district, hormozgan province, iran.

BACKGROUND: A successful malaria elimination program calls for enough attention to parasite carriers, especially asymptomatic malaria, as well as the diagnosis and treatment of clinical cases. Asymptomatic malaria is an infection that patients do not show any symptom; thus, these patients play critical role in the concept of an elimination program. The current investigation was conducted to evaluate the presence of these cases in Bashagard District, formerly a high malaria transmission area in Hormozgan Province, Iran. METHODS: Blood samples (n = 500) were collected from symptomless individuals residing in Bashagard to evaluate Plasmodium infection by using microscopic, serological and nested-PCR techniques. RESULTS: Regarding the microscopic and nested-PCR analysis, no asymptomatic infection was detected among studied individuals. Totally, 1% of the studied population (5 of 500) had anti PvMSP-1(19)-specific IgG antibody; however, only 0.2% (1 of 500) of the individuals was seropositive to recombinant PfMSP-1(19), using ELISA. CONCLUSION: This study showed no asymptomatic malaria infection in the studied population; hence malaria elimination is feasible and can be successfully carried out in this region.

Mice orally immunized with a transgenic plant expressing the glycoprotein of Crimean-Congo hemorrhagic fever virus.

While Crimean-Congo hemorrhagic fever (CCHF) has a high mortality rate in humans, the associated virus (CCHFV) does not induce clinical symptoms in animals, but animals play an important role in disease transmission to humans. Our aim in this study was to examine the immunogenicity of the CCHFV glycoprotein when expressed in the root and leaf of transgenic plants via hairy roots and stable transformation of tobacco plants, respectively. After confirmatory analyses of transgenic plant lines and quantification of the expressed glycoprotein, mice were either fed with the transgenic leaves or roots, fed the transgenic plant material and injected subcutaneously with the plant-made CCHFV glycoprotein (fed/boosted), vaccinated with an attenuated CCHF vaccine (positive control), or received no treatment (negative control). All immunized groups had a consistent rise in anti-glycoprotein IgG and IgA antibodies in their serum and feces, respectively. The mice in the fed/boosted group showed a significant rise in specific IgG antibodies after a single boost. Our results imply that oral immunization of animals with edible materials from transgenic plants is feasible, and further assessments are under way. In addition, while the study of CCHF is challenging, our protocol should be further used to study CCHFV infection in the knockout mouse model and virus neutralization assays in biosafety level 4 laboratories.

Immune responses elicited by co-immunization of Plasmodium vivax and P. falciparum MSP-1 using prime-boost immunization strategies.

Carboxy-terminus of merozoite surface protein-1 (MSP-1(19) ) is the major protein on the surface of the plasmodial merozoite that acts as one of the most important blood-stage vaccine candidates. The present investigation was designed to evaluate the immune responses when either two recombinant antigens (rPvMSP-1(19) + rPfMSP-1(19)) or two plasmid constructs (pcDNA3.1 hygro-PvMSP-1(19) + pcDNA3.1 hygro-PfMSP-1(19)) were administered in combination at a single site in mice by using different immunization strategies (protein/protein, DNA/DNA and DNA/protein) at weeks 0, 5 and 8. All mice were monitored for the level of MSP-1(19) -specific antibody for up to 40 weeks. The inclusion of both recombinant antigens in a vaccine mixture could not inhibit induction of antibodies to the other antigen when the two recombinant antigens were combined in immunization formulation. Interestingly, antisera from immunized mice with either recombinant antigen failed to cross-react with heterologous antigen. Moreover, the results of this study showed that co-immunization with both antigens at a single site generated a substantial PvMSP-1(19) - and PfMSP-1(19) -specific antibody responses and also IFN-γ cytokine production (Th1 response) in DNA/protein prime-boost immunization strategies. The increased humoral response to PvMSP-1(19) and PfMSP-1(19) lasted nearly a year after immunization. Therefore, the results of this study are encouraging for the development of multi-species malaria vaccine based on MSP-1(19) antigen.

Molecular and biomorphometrical identification of ovine babesiosis in iran.

BACKGROUND: Ovine babesiosis is the most important haemoparasitic tick-borne disease of small ruminants in Iran caused by Babesia ovis, B. motasi, and B. crassa. The aim of this study was to characterize the species of ovine Babesia species isolated from different geographical region of Iran. METHODS: One hundred fifty four blood samples collected from animals, which demonstrated the pale mucous membranes or hyperthermia. The specimens were transferred to the laboratory and the blood smears stained with Geimsa, the morphological and biometrical data of parasite in any infected erythrocyte have been considered. Extracted DNA from each blood samples were used in PCR and semi nested- PCR in order to confirm the presence of the species. RESULTS: Microscopical observation on 154 blood smears determined 38 (24.67%) and 40 (26%) samples were infected by Babesia and Theileria respectively. The mixed infections occurred in four (2.6%) samples. The results of the PCR assays showed nine (5.85%), 81 (53%) and 18 (11.7%) were distinguished as Babesia, Theileria and mixed infection, respectively. Semi nested- PCR did not confirm the presence of B. motasi. CONCLUSION: The causative organism of many cases of haemoprotozoal diseases, which recorded in previous studies, could be B. ovis or Theileria lestoquardi. The result confirmed that B. ovis was only species which causes babesiosis in the study areas. It seems that the biometrical polymorphisms could exist in B. ovis in Iran. This polymorphism could be a main problem in differentiation between B. ovis and B. motasi and it could be dissolved by specific PCR analysis.

First record of a new member of Anopheles Hyrcanus Group from Iran: molecular identification, diagnosis, phylogeny, status of kdr resistance and Plasmodium infection.

The current study aimed to provide further evidence on the status of species composition, insecticide resistance, and vectorial capacity within the members of Anopheles (Anopheles) Hyrcanus Group in Ardebil, Giulan, and Khuzestan provinces of Iran. Sequencing the internal transcribed spacer 2 (ITS2) of ribosomal DNA gene led to identification of two members of Hyrcanus complex: Anopheles hyrcanus Pallas and a new species/form, hereafter called Anopheles hyrcanus sp(IR) as a world record. Furthermore, we identified and compared partial sequences of exons I and II and the whole intron I region of insecticide resistance-related voltage-gated sodium channel (vgsc) gene in populations of Hyrcanus Group and other main old world Anopheles species. The ITS2 and vgsc sequences in members of Hyrcanus Group and other Anopheles species were used for construction of phylogenetic tree, which demonstrated the evolutionary relatedness among Western and Eastern Palearctic taxa within the Hyrcanus Group. A nested polymerase chain reaction assay for detection of Plasmodium species revealed the infection of Plasmodium falciparum within An. hyrcanus collected from Fooman district in Guilan province. The data from this study led to the introduction of a new member/form within the Hyrcanus Group, identification and definition of the status of knockdown resistance related to pyrethroids and DDT in their vgsc gene, detection of Plasmodium infection, and further evidence on genetic relatedness within these taxa. The overall results may suggest reconsidering the role ofAn. hyrcanus in malaria transmission, which would be useful for implementation and evaluation of malaria control programs in Western Palearctic region.

Quinoline resistance associated polymorphisms in the pfcrt, pfmdr1 and pfmrp genes of Plasmodium falciparum in Iran.

Plasmodium falciparum resistance to chloroquine (CQ) has been documented in Iran since the early 1980s and has since gradually increased. Iran is therefore reviewing its national drug policy for malaria control. We describe the prevalence of single nucleotide polymorphisms (SNP) associated with quinoline drug resistance in south eastern Iran. Pre-treatment blood from patients with uncomplicated but symptomatic P. falciparum infection was analysed. Polymorphisms at codons 76, 152, 163 and 220 of the pfcrt gene (chloroquine resistance transporter) and at codons 86, 184, 1034, 1042 and 1246 of the pfmdr1 gene (multidrug resistance) were determined by PCR-RFLP and sequencing. In addition, SNPs on a recently described multidrug resistance protein (pfmrp) and a microsatellite (MS-4760) in the pfnhe-1 (sodium hydrogen exchanger) gene associated with quinoline and quinine resistance, respectively, were investigated for the first time in field samples not from Thailand. pfcrt 76T was found in 99% and pfmdr1 86Y in 72% of the samples. pfmrp 191H and 437S associated with decreased quinoline response were found to be absolutely linked at a frequency of 13.6%. The pfnhe-1 MS-4760 one repeat allele associated to quinine response in vitro was also detected. Sequencing of the pfcrt 72-76 haplotype revealed that SVMNT was the most common allele as previously observed in India. This suggests that pfcrt found in the Iranian P. falciparum population may have the same origin as in the P. falciparum populations in India but different from that normally found in south east Asia. In conclusion, the frequencies of quinoline resistance associated gene polymorphisms in this region suggest a population that has been significantly selected for by the long use of CQ.

Molecular evidence of mixed P. vivax and P. falciparum infections in northern Islamic Republic of Iran.

This study compared basic microscopy with molecular detection of Plasmodium species. According to thick-film microscopy, 100% of 142 malaria cases in Pars-Abad, Ardebil province, were infected with a single species, P vivax. However, nested polymerase chain reaction (PCR) detected mixed species infections of both P. vivax and P. falciparum in 7.0%. In Mazanderan province, 2/20 blood films were diagnosed with only P. falciparum and 18/20 with only P. vivax. However, nested PCR detected 17/20, 2/20 and 1/20 with P. vivax only, P. falciparum only and mixed species respectively. The unexpected presence of P. falciparum urges prompt investigation and immediate treatment of malaria cases in this region.

IL-10 secretion and sensitivity in normal human intestine and inflammatory bowel disease.

Interleukin-10 (IL-10) deficiency in gene knockout mice causes chronic enterocolitis. We hypothesized that inflammation in human inflammatory bowel disease might result from innate alterations in the IL-10 pathway. Serum, supernatants, and mRNA of peripheral blood mononuclear cells (PBMC) and lamina propria mononuclear cells (LPMC) derived from inflamed (LPMC-i) and noninflamed colonic mucosa (LPMC-ni) were collected from patients with Crohn's colitis, ulcerative colitis, and controls. IL-10 protein concentrations and IL-10 mRNA were examined in response to PMA/CD3 or PHA stimulation. The response to rhIL-10 was assessed by inhibition of tumor necrosis factor-alpha (TNF-alpha), IL-6, and interferon-gamma (IFN-gamma) production. Serum IL-10 levels of inflammatory bowel disease (IBD) patients were within the normal range. IL-10 concentrations in supernatants from LPMC-i were significantly lower than from LPMC-ni or PBMC. No difference was seen between samples from ulcerative colitis and Crohn's disease. IL-10 mRNA was detected in 0/4 LPMC-i samples compared to 1/6 LPMC-ni and 6/6 PBMC. RhIL-10 inhibited TNF-alpha, IL-6, and IFN-gamma synthesis in PBMC. This effect was strongly diminished in LPMC. Disease-specific alterations were not detected. Our data suggest that LPMC derived from inflamed colonic mucosa have a reduced ability to produce and to respond to rhIL-10. A disease-specific alteration in the IL-10 pathway, however, was not found.

Effects of trovafloxacin on the IL-1-dependent activation of E-selectin in human endothelial cells in vitro.

E-selectin is an endothelial-specific surface protein, which is transiently expressed in response to inflammatory cytokines and plays an important role in the recruitment of leukocytes to the site of infection. The effect of two fluoroquinolones, ciprofloxacin (cipro) and trovafloxacin (trova), on the interleukin-1 (IL-1)-dependent activation of E-Selectin was studied on human umbilical vein endothelial cells (HUVEC) in vitro. Trova, at 80 microg/ml, affected the transient expression of E-selectin mRNA after pro-inflammatory stimulation with IL-1 leading to a sustained expression over 24 h. Surface expression of E-selectin remained upregulated after 24 h in a higher percentage of cells when they were activated in the presence of trova, as determined by flow cytometry analysis. Moreover, the concentration of shedded soluble E-selectin (sE-selectin) in the cell supernatant increased by 3.5 fold compared to those stimulated in the presence of cipro or without fluoroquinolones. Analogously, the antiproliferative effect of trova on endothelial cells was found to be more pronounced compared to cipro leading to an accumulation of cells arrested in G1-phase. These data provide evidence that accumulation of high concentration of trova in vivo in inflamed tissue might alter inflammatory responses.

Polar Plasmodium falciparum lipids induce lipogenesis in rat adipocytes in vitro.

Previous studies have shown that 'toxic malarial antigens' released by Plasmodium yoelii can induce hypoglycaemia in mice and act synergistically with insulin in stimulating lipogenesis in rat adipocytes in vitro. In this study, it was shown that similar bioactivity could be detected in Plasmodium falciparum culture supernatant, and the molecular basis of this activity was further investigated. Boiled spent culture medium from P. falciparum cultures ('BS-Pf') (exclusively released into the culture supernatant when schizonts rupture) acts in synergy with insulin to increase lipogenesis in a rat adipocyte assay by more than 250% (P < 0.001). Control preparations prepared from non-parasitized erythrocytes grown under similar conditions had no effect (P < 0.001). While contamination with mycoplasma has previously been shown to interfere with the interpretation of data obtained with other molecules thought to be released from P. falciparum in culture, including those inducing TNF-alpha and NO production by macrophages, such contamination was unequivocally ruled out here. BS-Pf alone did not stimulate the lipogenesis in short-term assays (less than 4 h), while long-term exposure of rat adipocytes to BS-Pf alone (12-24 h) caused a stimulation of lipogenesis at a level comparable to that observed with insulin. Furthermore, lipogenesis-inducing activity was also detected in the serum of squirrel monkeys infected with different species of malaria parasites (P. vivax, P. falciparum and P. brasilianum). Preliminary biochemical characterization showed that the biological activity was found in the solvent-extracted polar lipid fraction of boiled supernatant of P. falciparum cultures. All the different polar lipid fractions, collected from silica gel column chromatography, showed a comparable lipogenesis-inducing activity. Enzymatic treatment by phospholipase C of the lipid fraction, which co-migrated with the phosphatidylcholine standard, showed that the activity of the fraction was associated with the 1,2-diacylglycerol (1,2-DAG) moieties released from polar lipids. When this exogenous 1,2-DAG was added to the adipocyte cultures (short- and long-term cultures), it induced stimulation of lipogenesis in rat adipocytes, while no lipogenic activity was obtained from bacterial polar lipids and 1,2-DAG isolated from unparasitized erythrocytes. The importance of these findings is discussed with reference to other toxic malarial antigens and also to the potential role of these molecules in the induction of hypoglycaemia in the severe forms of malaria.

A novel high-purity isolation method for human peripheral blood neutrophils permitting polymerase chain reaction-based mRNA studies.

In the past few years, the role of polymorphonuclear neutrophils (PMN) in specific immune responses has gained significance due to their ability to express a variety of immunoregulatory molecules. However, controversial results concerning the potential of neutrophils for cytokine production have been obtained by sensitive molecular biological techniques. This problem might be related to contaminating leukocytes in conventionally isolated neutrophil suspensions as outlined by our study. We have established a novel method yielding highly purified neutrophils by combining a discontinuous Percoll gradient with fluorescence activated cell sorting of CD16bright cells. The latter step exploits the exceptionally high expression of Fc gammaRIIIB on PMN. Neutrophils could be enriched to homogeneity (> 99.9%) with a viability exceeding 90%. Contamination with NK cells or other lymphocytes, monocytes and eosinophils could be excluded as evaluated by reverse transcription-polymerase chain reaction (RT-PCR) with primers for HLA-DR, c-fms and CD52. The transcriptional potential of such purified neutrophils was confirmed by their ability to express MHC class II molecules after stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF). Our method should permit studies of PMN at the mRNA level and future investigations concerning the specificity of immunoregulatory molecule synthesis by neutrophils.

Lipopolysaccharide and interleukin 1 augment the effects of hypoxia and inflammation in human pulmonary arterial tissue.

The combined effects of hypoxia and interleukin 1, lipopolysaccharide, or tumor necrosis factor alpha on the expression of genes encoding endothelial constitutive and inducible nitric oxide synthases, endothelin 1, interleukin 6, and interleukin 8 were investigated in human primary pulmonary endothelial cells and whole pulmonary artery organoid cultures. Hypoxia decreased the expression of constitutive endothelial nitric oxide synthase (NOS-3) mRNA and NOS-3 protein as compared with normoxic conditions. The inhibition of expression of NOS-3 corresponded with a reduced production of NO. A combination of hypoxia with bacterial lipopolysaccharide, interleukin 1 beta, or tumor necrosis factor alpha augmented both effects. In contrast, the combination of hypoxia and the inflammatory mediators superinduced the expression of endothelin 1, interleukin 6, and interleukin 8. Here, we have shown that inflammatory mediators aggravate the effect of hypoxia on the down-regulation of NOS-3 and increase the expression of proinflammatory cytokines in human pulmonary endothelial cells and whole pulmonary artery organoid cultures.