First case report of paragonimiasis in a Malaysian man

@#Paragonimiasis is an infection caused by Paragonimus, a lung fluke and is acquired by eating raw or undercooked crustaceans containing the infective metacercariae. Herein, we report a case of paragonimiasis in a Malaysian man who presented with incidental findings from chest radiographs. Examination of his biopsied lung tissue and sputum specimen revealed Paragonimus sp. eggs, whereas stool examination showed the presence of Giardia cysts. Patient was succesfully treated with praziquantel and metronidazole respectively.

First case report of paragonimiasis in a Malaysian man.

Paragonimiasis is an infection caused by Paragonimus, a lung fluke and is acquired by eating raw or undercooked crustaceans containing the infective metacercariae. Herein, we report a case of paragonimiasis in a Malaysian man who presented with incidental findings from chest radiographs. Examination of his biopsied lung tissue and sputum specimen revealed Paragonimus sp. eggs, whereas stool examination showed the presence of Giardia cysts. Patient was succesfully treated with praziquantel and metronidazole respectively.

Efficacy of C.E.R.A. in Routine Clinical Practice for Correction of Anaemia and Maintenance of the Haemoglobin Levels in CKD Patients not on Dialysis.

INTRODUCTION: C.E.R.A. reported effective correction of anaemia and was well tolerated in International studies on CKD patients not on dialysis. OBJECTIVE: The study aimed to describe the management of renal anaemia in CKD patients not on dialysis with C.E.R.A. in routine clinical practice in India. METHODS: This was a prospective, single-arm, open-label, multi-centre, non-interventional, Phase IV study which followed 108 CKD Stage III-IV patients, not on dialysis with Hb < 10 g/dL for correction of anaemia with C.E.R.A. RESULTS: Of the 108 patients with Hb < 10 g/dL at baseline, 83 (90.2%) patients achieved target Hb of 10-12 g/dL and the time taken to achieve correction of anaemia was 9.6 weeks ± 6.13 weeks in the Intent-to-treat population. Haemoglobin concentration increased from 8.59 ± 0.808 g/dL pre-therapy to 10.91 ± 0.634 g/dL post-therapy. The change in mean ± SD Hb value was 2.32 ± 0.174 g/dL. Maintenance of Hb levels within the target range of Hb 10 - 12 g/dL was observed in 78.2% of ITT and 80.8% of the PP population for mean duration of 16.69 weeks. Four patients (3.7%) experienced 5 AEs and 2 patients (1.9%) experienced 3 SAEs in the safety population. As per the treating physician none of the AEs or SAEs was considered related to study drug. There were no deaths reported. CONCLUSIONS: This study demonstrated successful correction of anaemia in Indian patients with C.E.R.A. treatment as well as maintenance of Hb levels within the target range. C.E.R.A. was well tolerated with no new safety concerns specific to the Indian population. The less frequent up to monthly dosing schedule of C.E.R.A. may offer clinicians and patients a simplified regimen of anaemia management as compared to traditional frequently administered (thrice weekly to once weekly) ESAs.

Retraction Note to: Plant regeneration in Chlorophytum borivilianum Sant. et Fernand. from embryogenic callus and cell suspension culture and assessment of genetic fidelity of plants derived through somatic embryogenesis.

[This retracts the article DOI: 10.1007/s12298-012-0113-y.].

Hairy root biotechnology--indicative timeline to understand missing links and future outlook.

Agrobacterium rhizogenes-mediated hairy roots (HR) were developed in the laboratory to mimic the natural phenomenon of bacterial gene transfer and occurrence of disease syndrome. The timeline analysis revealed that during 90 s, the research expanded to the hairy root-based secondary metabolite production and different yield enhancement strategies like media optimization, up-scaling, metabolic engineering etc. An outlook indicates that much emphasis has been given to the strategies that are helpful in making this technology more practical in terms of high productivity at low cost. However, a sequential analysis of literature shows that this technique is upgraded to a biotechnology platform where different intra- and interdisciplinary work areas were established, progressed, and diverged to provide scientific benefits of various hairy root-based applications like phytoremediation, molecular farming, biotransformation, etc. In the present scenario, this biotechnology research platform includes (a) elemental research like hairy root-mediated secondary metabolite production coupled with productivity enhancement strategies and (b) HR-based functional research. The latter comprised of hairy root-based applied aspects such as generation of agro-economical traits in plants, production of high value as well as less hazardous molecules through biotransformation/farming and remediation, respectively. This review presents an indicative timeline portrayal of hairy root research reflected by a chronology of research outputs. The timeline also reveals a progressive trend in the state-of-art global advances in hairy root biotechnology. Furthermore, the review also discusses ideas to explore missing links and to deal with the challenges in future progression and prospects of research in all related fields of this important area of plant biotechnology.

DG-RAR for the treatment of symptomatic grade III and grade IV haemorrhoids: a 12-month multi-centre, prospective observational study.

BACKGROUND: Ultrasound-guided techniques represent a new treatment option in the treatment of haemorrhoids. Doppler-guided haemorrhoidal artery ligation (DG-HAL) proved efficacious in early haemorrhoidal disease, but lacks efficacy for stages III/IV. For these patients, haemorrhoidal artery ligation (HAL) has been combined with a running suture to reduce prolapsing haemorrhoidal tissue (recto-anal repair (RAR)). METHODS: A prospective observational study was conducted in 184 patients with grade III (58 %) or grade IV (42 %) haemorrhoids in seven coloproctological centres. Primary endpoints were the recurrence of symptoms and need of further treatment (medical or surgical). RESULTS: Post-operative complications were seen in 8 % of patients. After a follow-up of 3 months, 91 % of patients were free of symptoms and 91 % of patients were satisfied with the result. After a follow-up of 12 months, 89 % of patients were free of symptoms and 88 % were satisfied with the result. Nineteen per cent of patients received further medical or surgical treatment. CONCLUSIONS: Doppler-guided recto-anal repair (DG-RAR) proves to be an effective treatment option for the treatment of advanced haemorrhoidal disease that shows equal results to other established treatment options.

Efficiency of neural network-based combinatorial model predicting optimal culture conditions for maximum biomass yields in hairy root cultures.

KEY MESSAGE : ANN-based combinatorial model is proposed and its efficiency is assessed for the prediction of optimal culture conditions to achieve maximum productivity in a bioprocess in terms of high biomass. A neural network approach is utilized in combination with Hidden Markov concept to assess the optimal values of different environmental factors that result in maximum biomass productivity of cultured tissues after definite culture duration. Five hidden Markov models (HMMs) were derived for five test culture conditions, i.e. pH of liquid growth medium, volume of medium per culture vessel, sucrose concentration (%w/v) in growth medium, nitrate concentration (g/l) in the medium and finally the density of initial inoculum (g fresh weight) per culture vessel and their corresponding fresh weight biomass. The artificial neural network (ANN) model was represented as the function of these five Markov models, and the overall simulation of fresh weight biomass was done with this combinatorial ANN-HMM. The empirical results of Rauwolfia serpentina hairy roots were taken as model and compared with simulated results obtained from pure ANN and ANN-HMMs. The stochastic testing and Cronbach's α-value of pure and combinatorial model revealed more internal consistency and skewed character (0.4635) in histogram of ANN-HMM compared to pure ANN (0.3804). The simulated results for optimal conditions of maximum fresh weight production obtained from ANN-HMM and ANN model closely resemble the experimentally optimized culture conditions based on which highest fresh weight was obtained. However, only 2.99 % deviation from the experimental values could be observed in the values obtained from combinatorial model when compared to the pure ANN model (5.44 %). This comparison showed 45 % better potential of combinatorial model for the prediction of optimal culture conditions for the best growth of hairy root cultures.

ISSR and RAPD based evaluation of genetic stability of encapsulated micro shoots of Glycyrrhiza glabra following 6 months of storage.

In vitro grown axillary micro shoots of Glycyrrhiza glabra were encapsulated in alginate beads. Following 6 months of normal storage at 25 ± 2°C the re growth of encapsulated G. glabra micro shoots, reached 98% within 30 days of incubation on MS medium supplemented with 0.1 mg/l IAA. Re growth was characterized by the development of both shoot and root from single encapsulated micro shoot. Healthy plants were established to glass house with 95% survival. The genetic fidelity of plants obtained after conversion of alginate beads was ascertained through 10 RAPD and 13 ISSR primers. Of the 10 RAPD primers tested, 6 of them produced 14 clear and reproducible amplicons with an average of 2.3 bands per primer out of which 28.57% were polymorphic generated by only two primers. Eight ISSR primers produced total 37 bands ranging between 300 and 3,500 bp length. Number of scorable bands for each primer varied from 3 to 8 with an average of 4.6 bands per primer. Cluster analysis from ISSR and RAPD showed that all the tested plants including the mother plant distributed in two major groups with similarity coefficient ranging from 0.91 to 0.96 for RAPD and 0.89 to 0.97 for ISSR.

In vitro propagation of Rauwolfia serpentina using liquid medium, assessment of genetic fidelity of micropropagated plants, and simultaneous quantitation of reserpine, ajmaline, and ajmalicine.

Rauwolfia serpentina holds an important position in the pharmaceutical world because of its immense anti-hypertensive properties resulting from the presence of reserpine in the oleoresin fraction of the roots. Poor seed viability, low seed germination rate, and enormous genetic variability are the major constraints for the commercial cultivation of R. serpentina through conventional mode. The present optimized protocol offers an impeccable end to end method from the establishment of aseptic cultures to in-vitro plantlet production employing semisolid as well liquid nutrient culture medium and assessment of their genetic fidelity using polymerase chain reaction based rapid amplification of polymorphic DNA analysis. In vitro shoots multiplied on Murashige and Skoog basal liquid nutrients supplemented with benzo[a]pyrene (1.0 mg/L) and NAA (0.1 mg/L) and in-vitro rhizogenesis was observed in modified MS basal nutrient containing NAA (1.0 mg/L) and 2% sucrose. In-vitro raised plants exhibited 90-95% survival under glass house/field condition and 85% similarity in the plants regenerated through this protocol. Field established plants were harvested and extraction of indole alkaloid particularly reserpine, ajmaline and ajmalicine and their simultaneous quantitation was performed using monolithic reverse phase high-performance liquid chromatography (HPLC).

LC-ESI-MS analysis of taxoids from the bark of Taxus wallichiana.

LC-ESI-MS analysis was carried out for taxoid profiling of partially purified methanol extracts of the stem bark of Taxus wallichiana growing in different regions of the Himalayas (Kashmir, Himachal Pradesh, UP hills, Sikkim and Arunachal Pradesh). Cone voltage fragmentation of the protonated, ammonium or sodium cationized molecular species resulted in diagnostic fragment ions. Thus, information about the number and nature of substituents and the taxane skeleton (whether it is normal or rearranged) was readily available from the LC-ESI-MS spectra. The rearranged 11(15-->1)-abeo-taxanes showed a characteristic elimination of the hydroxyisopropyl along with an acetoxy group. The identification of the taxoids was achieved by comparison of the ESI mass spectra with those of the authentic taxoids available to us or by interpreting the ESI mass spectra. The results were also corroborated by MS/MS analysis of the partially purified extract injected directly into the ESI source. Paclitaxel, its analogues and their xylosides are present in samples from all the regions. An interesting observation is the detection of a large number of basic taxoids having nitrogen-containing side chains.

Phenobarbital, oxazepam and Wyeth 14,643 cause DNA damage as measured by the Comet assay.

Although phenobarbital, oxazepam and Wyeth 14,643 are carcinogens that do not form DNA adducts, they induce mutations in the Big Blue transgenic mouse model. The mutations produced by these compounds were predominantly G-->T and G-->C transversions that we suspect arose from oxidative damage to DNA. To test this, we employed the single cell electrophoresis (Comet) assay that detects alkali-labile lesions in cells sustaining DNA damage. Human myeloid leukemia K562 cells were treated with non-cytotoxic doses of the above compounds for 3 h, then placed on slides containing low melting point agarose. Cells were lysed, exposed to alkaline buffer, electrophoresed and analyzed by microscopy for the existence of DNA damage. Extensive DNA damage, most likely due to the existence of single- and double-strand breaks and apurinic/apyrimidinic (AP) sites, was observed in cells exposed to oxazepam (1 mM) and Wyeth 14,643 (0.5 mM). On the other hand, damage of this sort was not observed in cells exposed to phenobarbital (1 mM). However, the addition of S9 liver extracts to cells exposed in the presence of phenobarbital resulted in significant amounts of DNA damage. We conclude from these studies that two of the three compounds evaluated in this study mediate their mutagenic effects through oxidative stress, but that the mechanism of DNA damage caused by phenobarbital differs from that elicited by oxazepam and Wyeth 14,643.

Islet cell autoimmunity in youth onset diabetes mellitus in Northern India.

We characterised a consecutive cohort of 132 youth onset diabetic individuals (age at onset<30 years, mean duration of disease 5.5+/-6.0 years) from North India, by serological determination of the determination of the islet cell autoantibodies, GAD(65) and IA2, and clinically for coexisting autoimmune thyroid disease, malnutrition and pancreatic calcification. Five types of diabetes were delineated: Type 1 (37%), ketosis resistant (32%), Type 2 (13%), fibrocalculous pancreatopathy (11%) and autoimmune polyglandular syndrome (7%). C-peptide response to glucagon was assessed in a representative subset of 50 patients with Type 1, ketosis resistant, and autoimmune polyglandular syndrome. A total of 22.4% of Type 1 and 30% of autoimmune polyglandular syndrome subjects showed both GAD(65) plus IA-2 autoantibody positivity, significantly more than the 4.7% positivity shown by the ketosis resistant type. However, GAD(65) antibody positivity alone was seen in 38% of ketosis resistant subjects which was significantly more than the 14.2 and 10% positivity seen in Type 1 and autoimmune polyglandular groups, respectively. The fibrocalculous pancreatopathy group showed GAD(65) plus IA-2 autoantibody positivity in 14.2% and GAD(65) autoantibody alone positivity in 7.1%. 26 and 60%, respectively, of the Type 1 and autoimmune polyglandular syndrome groups had thyroid microsomal autoantibody positivity. Type 1 showed significantly less C-peptide response to glucagon when compared to the ketosis resistant and autoimmune polyglandular syndrome groups. The controls and Type 2 diabetic individuals tested negative for islet cell autoimmunity markers. These findings demonstrate a role of islet cell autoimmunity in the pathogenesis of four out of the five clinical types of youth onset diabetes seen in North India.

Autoimmune hypogonadism as part of an autoimmune polyglandular syndrome.

The most compelling case for autoimmune mediated hypogonadism occurs when ovarian failure is part of an autoimmune polyglandular syndrome (APS). In patients with the rare, recessively inherited type 1 APS (APS-1), characterized by the triad of chronic mucocutaneous moniliasis, hypoparathyroidism, and Addison's disease, primary amenorrhea (elevated pituitary gonadotropins) or oligomenorrhea and infertility are constant features. Ovarian failure is associated with autoantibodies to steroid hormone secreting cells in the adrenal cortex, Leydig cells of the testes, granulosa/thecal cells of the Graffian follicles, corpus luteum, and the syncytiotrophoblast of the placenta. These autoantibodies react with 3 P450 enzymes involved with steroidogenesis, namely, 21-hydroxylase (adrenal specific), 17 alpha-hydroxylase, and the side chain cleavage enzyme. Recently the 14 exon, APS-1 (autoimmune regulator or AIRE) gene has been cloned (chr. 21p22.3), and multiple mutants discovered. Parents who are obligatory heterozygotes for a single mutant gene lack clinical features of APS-1. They also do not develop APS-1 autoantibodies. Thus, hypogonadal patients without features of APS-1 are unlikely to have AIRE gene mutations. In the more common APS-2/3, characterized by combinations of autoimmune thyroid disease, immune mediated type 1 diabetes, vitiligo, pernicious anemia, and Addison's disease (type 2, not type 3), ovarian disease may be seen. In primary hypogonadism outside of the context of an APS, these autoantibodies are rare.

Diabetes vaccines: a future to be realized.

Immune-mediated (type 1) diabetes mellitus (IMD) is an autoimmune disease resulting from the chronic destruction of pancreatic islet cells by autoreactive T lymphocytes. Although there has been much advancement in diabetes management, targeting the precise etiology of the disease process has remained elusive. Recent progress in the understanding of the immunopathogenesis of IMD, however, has led to new intervention strategies, especially antigen-based therapies given as altered peptide ligands (APLs) or as vaccines. Instead of using immunosuppressive agents to suppress an already dysfunctional immune system, antigen specific vaccines or even non-antigen specific immunostimulants present a unique opportunity to boost regulatory function and thereby regain tolerance to self. We discuss here the pathogenesis of IMD as it relates to therapeutic possibilities, review various intervention strategies that have been successful in rodent models, and then present recent progress in human trials of diabetes intervention and prevention through vaccine prototypes.

Current cases in which epitope mimicry is considered as a component cause of autoimmune disease: immune-mediated (type 1) diabetes.

Autoimmune diseases result from a combination of genetic, immunologic, hormonal, and environmental factors. Infectious agents may induce the breakdown of immunological tolerance and the appearance of autoreactivity. However, the specific relationship between infection and autoimmunity is still unclear. One of the mechanisms responsible could be molecular mimicry between the infectious agent and self. The concept of molecular mimicry is a viable hypothesis in the investigation of the etiology, pathogenesis, treatment, and prevention of autoimmune disorders. Immune-mediated (type 1) diabetes in humans and in non-obese diabetic (NOD) mice is polygenic and characterized by autoimmune destruction of insulin-producing pancreatic beta cells in islets of Langerhans. In NOD mice, a T-helper 1 (Th1)-based autoimmune response arises spontaneously against glutamate decarboxylase (GAD) concurrently with the onset of insulitis. Subsequently. this Th1-type autoreactivity spreads intra- and intermolecularly to other beta cell autoantigens, suggesting that a Th1-type response is responsible for the progression of the disease, whereas Th2 responses when experimentally induced are protective. In humans, a homology between GAD and the P2-C protein of Coxsackie B make a cause-and-effect molecular mimicry an attractive hypothesis. Evidence to support the concept of molecular mimicry in diabetes is reviewed.

The human leukocyte antigen HLA DRB3*020/DQA1*0501 haplotype is associated with Graves' disease in African Americans.

Information on genetic susceptibility to Graves' disease in African Americans is limited. We studied DRB1, DQB1, DRB3 subtypes, DQA1*0501, DQA1*0201, and CTLA-4 polymorphisms in 49 African American patients with adult onset Graves' disease and 47 racially-matched controls using PCR-based sequence-specific priming methods. There were no significant differences in DRB1 or DQB1 allelic frequencies or CTLA-4 polymorphisms between patients and controls. However, we found that the frequency of DRB3 was significantly increased in the patients (75.5% vs. 57.4%, P = 0.006, X2 = 3.52), especially for the DRB3*0202 subtype (53.1% vs. 23.4, P = 0.003, X2 = 8.91). In this one respect, the finding was in concordance with our previous observations in Caucasian patients with adult-onset Graves' disease. In addition, whereas the frequency of DQA1*0501 was increased (P = 0.018, X2 = 5.63) in our patients, the haplotype of DRB3/DQA1*0501, or DRB3*0202/DQA1*0501 was found to be more strongly associated (P = 0.008, X2 = 7.0; P = 0.0008, X2 = 11.34, respectively). These data suggest that DRB3*0202, particularly when found with DQA1*0501 in a haplotype is a susceptible gene(s) for Graves' disease in adult African Americans. Considering these data with those in Caucasian patients, our results would suggest that the primary Graves susceptible locus is likely DRB3 and not DRB1.

Autoimmunity and diabetes.

The face of immune-mediated (type 1) diabetes is changing. No longer considered a disease confined to childhood, the incidence rate in Western countries is clearly rising and affecting younger children. Such a secular trend can only be explained on the basis of increased contacts with adverse environmental factors acting on a background of complex genetics. Multiple defects in immunological tolerance to "self' predispose to immune-mediated (type 1) diabetes. Initiation of immune responses involves the cytokine rich natural killer T cells. Such cells appear deficient in both humans and the rodent models of the disease. Furthermore, the regulatory abilities of T cells in general seem to be compromised. Effector mechanisms probably are dominated by cell-mediated beta cell destruction through apoptosis induction. Surprisingly, the essential antigen-presenting cells in the autoimmune processes involved appear to be B lymphocytes. The improved understanding of the beta cell autoantigens involved has led to better disease prediction. The long prodromal phase now readily identifiable through autoantibodies is spawning hopes of disease prevention, notably through antigen-based interventions or diabetes "vaccines."

Analysis of taxol and major taxoids in Himalayan yew, Taxus wallichiana.

A reversed-phase column liquid chromatography method for the analysis of taxol, 10-deacetylbaccatin III, baccatin IV, 1-hydroxybaccatin I, 2-acetoxybrevifoliol, brevifoliol, 2'-deacetoxydecinnamoyltaxinine J and 2'-deacetoxytaxinine J in yew needles has been developed using a Nova-Pak Phenyl column and a binary gradient profile. The various aspects of analysis such as extraction efficiency, detection limits, reproducibility and peak purity were validated using UV-Vis as well as photodiode array detection.

Protective effects of equine herpesvirus-1 (EHV-1) glycoprotein B in a murine model of EHV-1-induced abortion.

In an attempt to determine if pregnant mice could be protected from abortion subsequent to challenge with equine herpesvirus-1 (EHV-1) in the mouse model of EHV-1 disease, female BALB/c mice were inoculated with baculovirus-expressed EHV-1 glycoprotein B (bac-gB), wild-type baculovirus (bac-wt), rabbit kidney (RK-13) or baby hamster kidney (BHK-21) cells. Using an ELISA, antibodies against EHV-1 were detected in the serum of mice following two injections of bac-gB and were enhanced by a third injection, after which low levels of neutralising antibody were also detected. After mating, mice in the bac-gB, bac-wt and RK-immunised groups were infected intranasally with 10(7) pfu of EHV-1 on day 16 of pregnancy. All challenged mice experienced body weight loss post-infection (pi). However, postnatally, the gB-immunised group demonstrated body weight gain which was not seen in the other groups. There were no maternal deaths in the gB-immunised group but 1/6 bac-wt-immunised and 3/6 RK-immunised mice died post-challenge. Litter survival rate was significantly higher (p < 0.001) for the gB-immunised dams (54%) than that of either the bac-wt-(9%) or RK-immunised (0%) dams and the mean body weight of young from the surviving bac-wt-immunised litter was significantly (p = 0.021) lower than either the gB-immunised group or the BHK-immunised unchallenged group at 10 days of age. The virus was not isolated from any foetus from a gB-immunised dam. However, the virus was detected in 9% of foetuses from bac-wt-immunised and 21% of foetuses from RK-immunised dams.

Study of the protective immunity of co-expressed glycoprotein H and L of equine herpesvirus-1 in a murine intranasal infection model.

Equine herpesvirus-1 (EHV-1) glycoproteins H, and L (gH and gL) expressed individually or co-expressed by recombinant baculoviruses were used to immunise BALB/c mice prior to intranasal challenge in a murine model of respiratory infection. Only the co-expressed material (EHV-1 gH/gL) induced neutralising antibody (low levels). The same immunogen also produced the strongest cellular responses. Immunisation with gH/gL and, to a lesser extent, with gH alone was associated with a reduction of virus load in nasal turbinates and olfactory bulbs after challenge infection. Viraemia, detected by polymerase chain reaction, was also reduced. No such protective effects were observed for gL alone. Adoptive transfer of lymphocytes from gH/gL-immunised mice to näive mice subsequently challenged with EHV-1 indicated that both CD4+ and CD8+ cells had a role in protective immunity. Although clearance of EHV-1 from respiratory tissue was not as effective as previously found for glycoproteins D or C, these experiments provide evidence that the co-expression of EHV-1 gL with gH generates a conformational neutralising epitope which is not present in either molecule alone, and suggests that gH/gL antigen may have a better potential as a component of an EHV-1 vaccine than gH alone.