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Idiopathic pulmonary haemosiderosis is a rare disorder, with recurrent life-threatening alveolar haemorrhages and chronic lung parenchymal changes. It is associated with a triad of haemoptysis, iron deficiency anaemia, and diffuse pulmonary infiltrates. Although most cases are idiopathic, secondary haemosiderosis linked to known diseases has also been observed. Most of the cases remain undiagnosed because the disease is very low on the list of differentials. There is no specified age for the disease. The present study reports on an adolescent female patient who presented with microcytic anaemia and bilateral lung infiltrates to the National Institute of Child Health (NICH), Karachi, a tertiary care hospital. She was diagnosed with Idiopathic pulmonary haemosiderosis after ruling out other possibilities.

Cardiovascular diseases (CVDs) are the leading cause of mortality globally. Wald and Law proposed the idea of a "polypill"; a fixed dose combination therapy (FDC) in the form of a single pill to curb the CVD epidemic. Such a drug would include the combination of a broad spectrum of drugs including cholesterol lowering drugs, antihypertensive drugs, antiplatelet drugs, anticoagulation drugs, and antiarrhythmic drugs, which are frequently integrated to combat specific CVDs. This "polypill" holds the potential to pose several advantages like increased compliance, improved quality of life, risk factor control, psychological relief, and cost effectiveness along with minimal side effects. Several trials (like TIPS, UMPIRE, PolyIran, etc.) have tested different treatment strategies to test the hypothesis of Wald and Law. Unlike the past, physicians are now highly aware of this new strategy. The future of polypill in the management of CVD lies in a strategy where polypills are treated supplementary to the already existing preventive care, which includes lifestyle modifications and efforts to reduce tobacco use., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Fasciola gigantica is one of the aetiological trematodes associated with fascioliasis, which heavily impacts food-production systems and human and animal welfare on a global scale. In the absence of a vaccine, fascioliasis control and treatment is restricted to pasture management, such as clean grazing, and a limited array of chemotherapies, to which signs of resistance are beginning to appear. Research into novel control strategies is therefore urgently required and the advent of 'omics technologies presents considerable opportunity for novel drug and vaccine target discovery. Here, interrogation of the first available F. gigantica newly excysted juvenile (NEJ) transcriptome revealed several protein families of current interest to parasitic flatworm vaccine research, including orthologues of mammalian complement regulator CD59 of the Ly6 family. Ly6 proteins have previously been identified on the tegument of Schistosoma mansoni and induced protective immunity in vaccination trials. Incorporating the recently available F. gigantica genome, the current work revealed 20 novel Ly6 family members in F. gigantica and, in parallel, significantly extended the F. hepatica complement from 3 to 18 members. Phylogenetic analysis revealed several distinct clades within the family, some of which are unique to Fasciola spp. trematodes. Analysis of available proteomic databases also revealed three of the newly discovered FhLy6s were present in extracellular vesicles, which have previously been prioritised in studying the host-parasite interface. The presentation of this new transcriptomic resource, in addition to the Ly6 family proteins here identified, represents a wealth of opportunity for future vaccine research.

Fasciolosis an economically important global disease of ruminants in the temperate and tropical regions, caused by Fasciola hepatica and F. gigantica, respectively, also poses a potential zoonotic threat. In India alone it causes huge losses to stakeholders. Anthelmintics including triclabendazole have been used to control this menace but the emerging resistance against the available compounds necessitates identification of novel and alternative therapeutic measures involving plant derived natural compounds for their anthelmintic potential. Thymoquinone (T) and curcumin (C), the active ingredients of Nigella sativa and Curcuma longa respectively have been used as antiparasitic agents but the information on their flukicidal effect is very limited. Adult flukes of F. gigantica were in vitro exposed to different concentrations of thymoquinone and curcumin separately for 3h at 37+ 1°C. A significant (p<0.05) reduction in the worm motility at 60 μM concentration of both T and C was observed though all the worms remained alive after 3h exposure, whereas the effect on egg shedding was statistically insignificant. Pronounced tegumental disruptions and erosion of spines in the posterior region and around the acetabulum was evident. A significant (p<0.05) decrease in glutathione-S-transferase and superoxide dismutase activity and reduced glutathione (GSH) level was observed, while protein carbonylation increased differentially. A significant inhibition of CathepsinL (CatL) gene expression in thymoquinone treated worms was also evident. Further, in silico molecular docking of T and C with CatL revealed a stronger interaction of curcumin with the involvement of higher number of amino acids as compared to thymoquinone that could be more effective in inhibiting the antioxidant enzymes of F. gigantica. It is concluded that both the compounds understudy will decrease the detoxification ability of F. gigantica, while inhibition of CatL will significantly affect their virulence potential. Thus, both thymoquinone and curcumin appeared to be promising anthelmintic compounds for further investigations., Competing Interests: The authors have declared that no competing interests exist.

The liver flukes Fasciola hepatica and F. gigantica infect livestock worldwide and threaten food security with climate change and problematic control measures spreading disease. Fascioliasis is also a foodborne disease with up to 17 million humans infected. In the absence of vaccines, treatment depends on triclabendazole (TCBZ), and overuse has led to widespread resistance, compromising future TCBZ control. Reductionist biology from many laboratories has predicted new therapeutic targets. To this end, the fatty-acid-binding protein (FABP) superfamily has proposed multifunctional roles, including functions intersecting vaccine and drug therapy, such as immune modulation and anthelmintic sequestration. Research is hindered by a lack of understanding of the full FABP superfamily complement. Although discovery studies predicted FABPs as promising vaccine candidates, it is unclear if uncharacterized FABPs are more relevant for vaccine formulations. We have coupled genome, transcriptome, and EST data mining with proteomics and phylogenetics to reveal a liver fluke FABP superfamily of seven clades: previously identified clades I-III and newly identified clades IV-VII. All new clade FABPs were analyzed using bioinformatics and cloned from both liver flukes. The extended FABP data set will provide new study tools to research the role of FABPs in parasite biology and as therapy targets.

The digenetic trematodes, Fasciola gigantica and Gigantocotyle explanatum, belonging to the family Fasciolidae and Paramphistomidae respectively, have been often found to concurrently infect the liver of Indian water buffalo Bubalus bubalis, causing serious pathological damage to the vital organ, incurring huge economic losses. In the present study the soluble gene products of both F. gigantica and G. explanatum were analyzed by 2 dimensional polyacrylamide gel electrophoresis. The soluble proteomic profile revealed considerable similarity as well as differences in the size, distribution pattern, total number, the isoelectric point (pI) and molecular weight (Mr) of the resolved polypeptide spots. The maximum number of polypeptide spots with a molecular weight range of >10 to 160 kDa were recorded with a pI range of 7-9 followed by pI range of 5-7, 9-10 and 3-5 in both the parasites. However, considerable variation was recorded in the Mr of the polypeptides belonging to each pI range. The genetic heterogeneity could be an obvious contributing factor for such differences but some polypeptides appeared to be conserved in the two species. The molecular similarities and the habitat preference by these worms may be a consequence of microenvironmental cues that guide these flukes to reach their habitat through different routes and establish a successful host-parasite relationship.

Osteopetrosis is a rare hereditary disorder of osteoclast dysfunction leading to abnormally dense and sclerotic bones that are fragile and break easily. It can be inherited in various patterns like autosomal-dominant, autosomal-recessive or as X-linked traits, but the most grievous forms of its inheritance are the autosomal-recessive ones, which show early onset and are associated with very poor prognosis. We report here the case of an asymptomatic young boy, who was diagnosed as the case of autosomal recessive osteopetrosis on the basis of his genetic studies. The reason for his unusual asymptomatic disease was the location of mutation in TCIRG1 gene that was revealed from his genetic studies. Another unusual point about him was his survival at this age, which is surprisingly rewarding as patients with autosomal recessive osteopetrosis usually die earlier by the age of 2-3 years.

Helminth parasites of veterinary importance cause huge revenue losses to agrarian economy worldwide. With the emergence of drug resistance against the current formulations, there is a need to focus on the alternative approaches in order to control this menace. In the present study, biocompatible zinc oxide nanoparticles (ZnO NPs) were used to see their in vitro effect on the biliary amphistomes, Gigantocotyle explanatum, infecting Bubalus bubalis because these nanoparticles are involved in generation of free radicals that induce oxidative stress, resulting in disruption of cellular machinery. The ZnO NPs were synthesized by using egg albumin as a biotemplate and subsequently characterized by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), X-ray Diffraction and Spectrophotometrical, which showed that ZnO NPs were highly purified wurtzite type polycrystals, with a mean size of 16.7 nm. When the parasites were treated with lower concentrations (0.004% and 0.008%) of the ZnO NPs, the worms mounted a protective response by stimulating the antioxidant system but the treatment of G. explanatum with 0.012% ZnO NPs produced significant inhibition of the antioxidant enzymes like superoxide dismutase (SOD) (p< 0.05) and glutathione S- transferase (GST) (p<0.01), while the level of malondialdehyde (MDA), a lipid peroxidation marker, was significantly (p< 0.01) elevated. SEM and histopathology revealed pronounced tegumental damage showing the disruption of surface papillae and the annulations, particularly in the posterior region near acetabulum. The under expression of a number of polypeptides, loss of worm motility in a time dependent manner, further reflect strong anthelmintic potential of ZnO NPs. It can be concluded that the anthelmintic effect might be due to the production of reactive oxygen species that target a variety of macromolecules such as nucleic acid, protein and lipids which are involved in different cellular processes.

Parasitic helminths polarize immune response of their vertebrate hosts towards anti-inflammatory Th2 type and therefore it is hypothesized that they may suppress the inflammatory conditions in autoimmune disorders. The present study was undertaken to investigate in vivo immunomodulatory and therapeutic potential of somatic antigens (Ag) of liver infecting digenetic trematodes [Fasciola gigantica (Fg) and Gigantocotyle explanatum (Ge)] in collagen-induced arthritic (CIA) Wistar rats. The CIA rats were administered subcutaneously with different doses (50 μg, 100 μg and 150 μg) of somatic antigens of Fg and Ge, daily for 21 days, the time period required to establish infection in natural host (Bubalus bubalis). Thereafter, the control, diseased and treated rats were compared for different parameters viz. hind paw thickness; serum interleukins, IL-4 and IL-10, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ); expression level of matrix metalloproteinases (MMPs) -2, -9, -13 and nitric oxide (NO) in knee joints and patellar morphology. The CIA rats treated with different antigens, Fg-Ag and Ge-Ag, show significant amelioration of the disease by down regulation of serum TNF-α and IFN-γ (p< 0.05) and upregulation of IL-4 and IL-10 cytokines (p< 0.05); inhibition (p< 0.05) of MMPs (-2,-9,-13) and NO in knee joints and improved patellar morphology with decreased synovial hypertrophy and reduced infiltration of ploymorphonuclear cells. The activity of pro as well as active MMPs (-2 and -9) and active MMP-13 in knee joints of CIA rats was very high compared to the control and treatment groups, suggesting the extent of collagen degradation in CIA rats. Interestingly, the highest dose (150 μg) of Ge-Ag almost wiped out MMP-13 expression. The overall findings suggest that the somatic proteins of Ge-Ag appeared to be therapeutically more effective than Fg-Ag, reflecting interspecific molecular differences which could contribute to the ability of these worms to successfully ameliorate the pathology of CIA.

Triclabendazole (TCBZ), the anthelmintic drug active against both mature and immature liver flukes, was used to investigate the effect of in vivo treatment on the tegumental surface of juvenile Fasciola gigantica. Five goats were infected with 150 F. gigantica metacercariae each by oral gavage. Four of them were treated with single dose of TCBZ at 10mg/kg at four weeks post-infection. They were euthanized at 0 (untreated), 24, 48, 72 and 96h post treatment. Juvenile flukes were manually retrieved from the goat livers and processed for scanning electron microscopy. In control flukes, the anterior region was adorned with sharply pointed spines projecting away from the surface, while in the posterior region, spines become shorter and narrower, loosing serration and with the appearance of distinct furrows and papillae. The dorsal surface retained the same pattern of surface architecture similar to that of ventral surface. Flukes obtained from 24h post-treatment did not show any apparent change and were still very active. However, there were limited movements and some blebbing, swelling, deposition of tegumental secretions and some flattening displayed by the flukes of 48h post-treatment. All the worms were found dead 72h post-treatment and showed advanced level of tegumental disruptions, consisting of severe distortion of spines, sloughing off the tegument to expose the basal lamina, formation of pores and isolated patches of lesions. By 96h post-treatment, the disruption was extremely severe and the tegument was completely sheared off causing deeper lesions that exposed the underlying musculature. The disruption was more severe at posterior than anterior region and on ventral than dorsal surface. The present study further establishes the time-course of TCBZ action in vivo with 100% efficacy against the juvenile tropical liver fluke., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Clinostomum complanatum is a digenetic trematode that causes yellow grub disease in some fish species and also shows zoonotic potential by sporadically infecting humans. In this study, progenetic metacercariae of C. complanatum were obtained from the fish Trichogaster fasciatus, and were aseptically placed in conjunctival incisions made in the superior and inferior fornices of the eye of rabbits, which served as the experimental hosts. Worms were harvested without necropsy of the host on days 4 and 8 post infection, to observe in vivo transformation of the progenetic metacercariae into ovigerous adult worms. The worms appeared to cause minimal damage to the host although they were tenaciously attached. In vivo maturation was evident by the development of the vitellaria, enlargement of gonads, the presence of a large number of shelled eggs in a distended uterus and ramifications of the intestinal caeca. Obtaining mature ovigerous worms without sacrificing the host clearly gives the rabbit eye model an advantage over those described previously. Due to the relative advantage of the short time required for maturation and the prolific egg production by C. complanatum, it is suggested that this host-parasite system could be used as an excellent model for classroom teaching of trematode biology and to investigate the cues involved in in vivo transformation and host-parasite interactions.

Cysteine proteases of parasite organisms play numerous indispensable roles in tissue penetration, feeding, immunoevasion, virulence, egg hatching and metacercarial excystment. They are critical key enzymes in the biology of parasites and have been exploited as serodiagnostic markers, therapeutic and vaccine targets. In the present study, the cysteine proteases in the in vitro released excretory/secretory (E/S) products of the digenetic trematode parasite, Euclinostomum heterostomum have been analysed. The encysted progenetic metacercariae of E. heterostomum collected from the infected liver and kidney of Channa punctatus were excysted in vitro and incubated in phosphate buffer at 37 ± 1 °C, and the E/S products released were analysed. The spectrophotometric analysis of the proteases revealed active hydrolysis of chromogenic substrate, azocoll, in a time-, temperature- and pH-dependent manner. Optimum activity was observed at pH 7.0 at 37 ± 1 °C, and with 1 mM each of various protease inhibitors (Mini Protease Inhibitor Cocktail, ethylene diaminetetraacetic acid, phenyl methyl sulphonyl fluoride, iodoacetamide and 1,10-phenanthroline) used, significant inhibition was observed by iodoacetamide and 85% of inhibition at a concentration of 2 mM, suggesting that cysteine protease is a major component in the E/S of this parasite. Four discrete protease bands of Mr 36, 39, 43 and 47 kDa were identified by gelatin-substrate zymography. Maximum gelatinolytic activity was observed at pH 7.0, and among various inhibitors used, almost complete disappearance of protease bands was observed by 2 mM iodoacetamide. The proteolytic cleavage of bovine serum albumin, bovine haemoglobin and human haemoglobin in vitro were also studied.

Viability of eggs is important for the successful completion of trematode life cycle, both in natural and laboratory conditions. The present study was designed to check the viability of eggs released by the digenetic trematode parasite Clinostomum complanatum transformed in experimentally infected chicken and rabbit eye. The incubation of the released eggs in distilled water at 28 ± 1 °C led to the embryonation followed by hatching on tenth day to release miracidia. These can be used to infect the snails. We propose that these two in vivo model systems can be used as a source of viable eggs for further studies on developmental biology and life cycle where in law-protected animals are not to be used. To the best of our knowledge, in contrast to the previous attempts, this is the first successful study to report any experimental model to produce ovigerous adult worms capable of releasing viable eggs.

The infection of gastrointestinal helminths in livestock is routinely diagnosed by microscopical examination of faecal samples for the presence of ova/eggs but this approach becomes ineffective for the seasonally egg producing trematodes. Therefore, an alternative approach to detect the coproantigens of liver and rumen amphistomes, Gigantocotyle explanatum and Gastrothylax crumenifer respectively, infecting Indian water buffalo Bubalus bubalis, was undertaken using ELISA, immunodot and countercurrent immunoelectrophoresis (CCIEP). The hyperimmune polyclonal antisera were separately raised in rabbits against excretory/secretory (ES) antigens of both the flukes under study. An overall 70% buffalo faecal samples were tested positive for G. crumenifer and 75% for G. explanatum in Aligarh region. The ELISA results reflected higher infection intensity among individual buffaloes that was also observed at necropsy. Using the respective homologous hyperimmune antiserum, 55% buffaloes tested positive for G. crumenifer and 65% positive for G. explanatum in immunodot assay. Further, the faecal samples with high absorbance values in ELISA and strong immunodot reaction tested positive in CCIEP. The analysis of CCIEP result revealed two and one precipitin bands in G. crumenifer and G. explanatum respectively, indicating prominent antigenic differences in the coproantigens of these two parasites. Taken together, it is suggested that polyclonal antibodies could be conveniently used for the detection of coproantigens by ELISA and immunodot methods, particularly during the non-egg producing phase of the seasonally regulated reproductive cycle of the rumen amphistome G. crumenifer. It is concluded that the coproantigen detection is a good alternative over conventional method for the diagnosis of amphistomosis in livestock; however, further studies are required on a larger sample size of field buffaloes to augment the reproducibility of the present results., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Fasciolosis is an important foodborne, zoonotic disease of livestock and humans, with global annual health and economic losses estimated at several billion US$. Fasciola hepatica is the major species in temperate regions, while F. gigantica dominates in the tropics. In the absence of commercially available vaccines to control fasciolosis, increasing reports of resistance to current chemotherapeutic strategies and the spread of fasciolosis into new areas, new functional genomics approaches are being used to identify potential new drug targets and vaccine candidates. The glutathione transferase (GST) superfamily is both a candidate drug and vaccine target. This study reports the identification of a putatively novel Sigma class GST, present in a water-soluble cytosol extract from the tropical liver fluke F. gigantica. The GST was cloned and expressed as an enzymically active recombinant protein. This GST shares a greater identity with the human schistosomiasis GST vaccine currently at Phase II clinical trials than previously discovered F. gigantica GSTs, stimulating interest in its immuno-protective properties. In addition, in silico analysis of the GST superfamily of both F. gigantica and F. hepatica has revealed an additional Mu class GST, Omega class GSTs, and for the first time, a Zeta class member.

The levels of oxidative stress markers are an important indicator of the physiological state of the parasite and its host. In the present study levels of lipid peroxidation, glutathione S transferase, glutathione, superoxide dismutase and catalase were determined in the Clinostomum complanatum progenetic metacercaria, obtained from the fish peritoneum (a hypoxic habitat). The in vivo transformed ovigerous adult worms were obtained from the aerobic environment of the buccopharyngeal region of experimentally infected chickens. Levels of antioxidant molecules were also determined in the blood of experimentally infected chickens. An increase in the levels of lipid peroxidation, and a significant decrease in the levels of glutathione S transferase, glutathione, superoxide dismutase and catalase was observed in the infected host as compared to the controls. In the ovigerous worms, the levels of lipid peroxidation, glutathione S transferase, glutathione, superoxide dismutase were found to be significantly less than the levels observed in the progenetic metacercaria. Since the establishment of worm in the buccal cavity of the avian host would lead to its exposure to oxygen and the haematophagous nature of the parasite also exposes it to the free radicals in the host blood, the progenetic metacercaria has evolved to produce excess free radical scavenging molecules reserved to combat the oxidative stress encountered within the microhabitat of the definitive host., (Copyright © 2011 Elsevier B.V. All rights reserved.)

4-Aminobiphenyl, a known carcinogen, has many environmental sources like cigarette smoke, industrial waste, and so forth. It can be metabolized to form a potent mutagen, N-hydroxy-N-acetyl-4-aminobiphenyl (N-OH-AABP) that undergoes further processing to form electrophilic nitrenium ions which interact with DNA-forming covalent adducts, thereby exerting genotoxic effects. While the mutagenicity of N-OH-AABP has been amply reported, no extensive studies have been performed to assess the immunogenicity of N-OH-AABP-modified DNA. In this study, human placental DNA was modified with N-OH-AABP, and the structural perturbations in the DNA molecule were evaluated by ultraviolet spectroscopy and nuclease S1 digestion. Native and N-OH-AABP-modified DNA were used as antigens for immunizing female rabbits. The modified DNA was found to be highly immunogenic, eliciting high titer immunogen-specific antibodies, while the native form was almost nonimmunogenic. The induced antibodies exhibited wide range of heterogeneity in recognizing various nucleic acid conformers and DNA bases. We also detected deposits of immune complex in glomerular basement membrane in rabbits immunized with N-OH-AABP-DNA. Possible role of N-OH-AABP-DNA in the induction of antibodies in cancer patients and the related consequences have been discussed., (Copyright © 2012 Wiley Periodicals, Inc.)

The antimicrofilarial efficacy of Trachispermum ammi extacts in vitro and in vivo using Setaria cervi as a model, was investigated. T. ammi seed extracts were prepared using different solvents (with increasing order of polarity of the solvent) including petroleum ether, diethyl ether, chloroform, ethyl acetate, acetone, ethanol, and methanol. The extracts were tested for in vitro antimicrofilarial activity. The ethanolic and the methanolic extracts showed maximum activity in causing flaccidity in the microfilariae. The extracts were potent even at concentrations as low as 5 μl/ml. When orally administered to experimentally infected rats, the extracts eliminated circulating microfilariae within 2 weeks. It is inferred that the antimicrofilarial molecule(s), are polar in nature. They induce flaccidity in the microfilariae, by possibly inhibiting monoamine oxidase. This communication supplements the ethnopharmacological information for the use of T. ammi as an antihelminthic, and indicates that T. ammi could be used as a potential source of antimicrofilarial drugs.

The dramatic and spontaneous exodus of live Clinostomum complanatum progenetic metacercaria from the gill slits of the dying intermediate host, Trichogaster fasciatus is reported. Basic water parameter tests for dissolved oxygen, pH and temperature revealed slightly lower level of dissolved oxygen in tank water used for water change. To the best of our knowledge, it is the first report of a digenean metacercariae, en mass leaving their intermediate host, upon its death in search of an alternative host to support their survival and help in continuing their life cycle.

The present study was carried out to identify the excretory/secretory (E/S) antigens of the rumen infecting digenetic trematode Gastrothylax crumenifer that may be useful for the immunodiagnosis of rumen amphistomosis particularly during the pre-monsoon season during which this rumen parasite stops shedding eggs. The in vitro released E/S proteins were purified on a Sephadex G-200 column. The gel filtration profile revealed three distinct fractions F1-F3 where F1 and F3 appeared as sharp peaks while the F2 fraction was dispersed. The antibody titre against each of the purified E/S fractions was determined by ELISA using anti-whole E/S polyclonal antibodies raised in rabbit. Among the three fractions, the antibody titre against F1 was highest (1:12,800) whereas IgG titre was very low (1:50) for fraction F2 and F3 (1:100). Of the total polypeptides resolved on gradient SDS-PAGE, only a few antigenic polypeptides were detected in each fraction with hyperimmune anti-serum as revealed by Western Blot analysis. However, a 33 kDa antigen detected in each fraction appeared to be immunodominant which could be exploited for the diagnosis of the pouched amphistome., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Glutamate dehydrogenase (GLDH) (EC 1.4.1.3) is a ubiquitous enzyme, which is present at the protein and carbohydrate metabolism crossroads. The enzyme activity was investigated in biliary and rumen amphistomes, Gigantocotyle explanatum and Gastrothylax crumenifer, respectively, infecting the Indian water buffalo Bubalus bubalis. The enzyme activity was consistently higher in G. explanatum as compared to G. crumenifer, where NAD(H) was utilized as coenzyme and the pH optima was recorded at 8. The K(m) and V(max) values for α-ketoglutarate were 2.1 mM and 9.09 units in G. explanatum, whereas 3.03 mM and 1.90 units in G. crumenifer, respectively. Among the allosteric modulator nucleotides, AMP, ADP, ATP, GMP, CMP and UMP, only AMP enhanced GLDH activity in G. crumenifer while ADP was stimulatory in G. explanatum. The amino acid leucine stimulated the GLDH activity in both the amphistomes while alanine was stimulatory only in G. crumenifer. Pronounced interspecific differences in response to different metabolic inhibitors like diethyldithiocarbamate, semicarbazide hydrochloride and mercurial ions were also observed. The osmotic stress alters the enzyme activity, particularly in hypertonic saline the GLDH activity increased significantly (p < 0.01) in G. explanatum, while insignificant effects were observed in rumen dwelling G. crumenifer. Histoenzymology revealed region/tissue specific distribution of GLDH with prominent staining in tissues like vitellaria, lymph system and tegument/subtegument, thus showing specific distribution of GLDH indicating differential metabolic state. Such intergeneric differences in GLDH activity could also be a consequence of occupying different microenvironments within the same host.

CD166 is an Ig superfamily molecule that binds homotypically to itself and heterotypically to CD6. Interactions between CD6 and CD166 are important during immune development and in alloreactivity. CD166 is expressed at increased levels in selected cancers and in rheumatoid arthritis synovium. Knowledge that CD166 was expressed in normal human salivary epithelium led to these studies of CD166 and CD6 in diseased mouse salivary glands, that resemble pathology seen in the human disease, Sjögren's syndrome. We showed that in mouse salivary epithelium CD166 was expressed but that expression of CD166 did not necessarily predict its function. Recombinant soluble CD6-Ig bound to CD6 ligands (CD6L) on transformed and freshly isolated salivary epithelial cells. Cross-blocking studies showed that binding of CD6-Ig to salivary epithelium was in part dependent on CD166, but that CD6-Ig binding may also involve additional CD6L. Binding of CD6-Ig was sensitive to trypsin digestion but resistant to digestion by collagenase and sialidase. Anti-CD166 ab precipitated CD166 from salivary epithelium pre- and post-treatment with the pro-inflammatory cytokine IFN-gamma. In contrast CD6-Ig only precipitated CD166 from IFN-gamma treated cells. More extensive colocalization between CD166 and the actin cytoskeleton was observed in sialoadenitis epithelium compared to control. We conclude that during sialoadenitis, CD166 undergoes a gain of function, resulting in closer association with the actin cytoskeleton and increased capacity to bind CD6. We suggest that altered CD166 function may contribute to the pro-inflammatory milieu during sialoadenitis seen in Sjögren's syndrome.

The number of cases of treated end-stage renal disease (ESRD) attributable to type 2 diabetes and survival after the onset of renal replacement therapy was examined in the Commonwealth of the Northern Mariana Islands (CNMI). All Chamorros and Carolinians to receive renal replacement therapy for ESRD between January 1982 and December 2002 were identified. Changes in survival over time were examined by dividing the study into three equal periods. Of 180 new cases of ESRD, 137 (76%; 101 Chamorros, 36 Carolinians) were attributed to diabetes. Ninety-nine subjects, 80% of whom had diabetic ESRD, began renal replacement therapy in the last 7 years of the study compared with 81 (72% with diabetic ESRD) in the previous 14 years. All 137 of the diabetic subjects received haemodialysis. During the 21-year study period, 79 of the diabetic subjects receiving dialysis died. The median survival after the onset of haemodialysis was 37 months in the first time period (1982-1988), 47 months in the second period (1989-1995) and 67 months in the third period (1996-2002). The death rate in the first period was 4.3 times (95% CI, 2.1-8.9) as high and the second period was 2.9 times (95% CI, 1.5-5.8) as high as the most recent period, after adjustment for age, sex and ethnicity in a proportional-hazards analysis. The number of diabetic patients in CNMI who are receiving renal replacement therapy is rising rapidly. Considerable improvement in survival after the onset of haemodialysis has occurred over the past 21 years.

CD6 is a T cell surface glycoprotein that plays an important role in interactions of thymocytes with thymic epithelial cells and in mature T cell interactions with selected nonprofessional tissue APCs. We describe a novel CD6 ligand (CD6L) 3A11 Ag that is distinct from the known CD6L (CD166). The 3A11 protein is expressed on cells derived from human thymus, skin, synovium, and cartilage, and its expression is enhanced by IFN-gamma. mAbs directed against the 3A11 Ag and CD166 exhibit distinct patterns of binding to a panel of cell lines. Confocal microscopy shows that both CD166 and the 3A11 Ag are expressed at the cell surface, and that these proteins colocalize. The 3A11 Ag has a molecular mass of 130 kDa and is immunoprecipitated using either mAb 3A11 or soluble CD6-Ig fusion protein. mAbs directed against individual CD6L were less potent than was soluble CD6-Ig fusion protein in reducing adhesion of T cells to adherent 3A11-positive epithelial cells in vitro, suggesting that these Abs recognize epitopes on the 3A11 Ag and CD166 that are distinct from CD6 binding sites. Finally, transfection of epithelial cells with CD166-specific small interfering RNAs significantly decreased CD166 expression without alteration in 3A11 Ag levels, and thus confirmed that these two CD6L are distinct. Taken together, our data identifies a novel 130-kDa CD6L that may mediate interactions of synovial and epithelial cells with T lymphocytes.

A 5-year randomized, double blind, placebo-controlled study was carried out to determine the effect of the angiotensin-converting enzyme (ACE) inhibitor enalapril (E) on the progress of renal function and histology in subjects with type 1 diabetes and microalbuminuria. Seventy three type 1 diabetic patients with BP <140/90 and with persistent albuminuria (AER 20-200 microg/min) and normal renal function were randomly assigned to receive E (n=37) or placebo (n=36). A percutaneous renal biopsy was successfully performed in 69 patients and repeated in 59 patients after 5 years. The mean glomerular volume (MGV), mesangial volume (Vv mes) and glomerular basement membrane thickness (GBMT) were measured histomorphometrically. Before treatment, both groups had similar clinical characteristics, blood pressure, HbA(1c), albumin excretion rate (AER), glomerular filtration rate (GFR), serum creatinine and renal structural damage. Blood pressure was well controlled in both groups. In the patients treated with E, albuminuria decreased significantly (P<0.05) and only 8.1% (3/37) of subjects progressed to clinical albuminuria (AER >300 mg/24 h) compared with 30.5% (11/36) in the placebo group. The E treatment resulted in absolute risk reduction of 22.4 percentage points for the development of clinical albuminuria over a 5-year period (P<0.01). After 5 years of treatment, GBM thickness showed a consistent, though statistically insignificant, increase in the placebo group, whereas it remained stable in the E group. A significant increase in MGV and Vv mes was also observed in the placebo group on completion of the study. The present study indicates that long term therapy with E may decrease or delay the progression of structural glomerular damage in microalbuminuric diabetic subjects without marked hypertension (BP <140/90).

The quantitative assay of mitochondrial monoamine oxidase (MAO) activity revealed a higher enzyme level in Explanatum explanatum than Gastrothylax crumenifer. The specific MAO inhibitors, chlorgyline, pargyline, deprenyl and nialamide produced different degrees of interspecific inhibition. The differential effects on enzyme activity of chlorgyline and deprenyl suggests the possible existence of polymorphic forms of the enzyme, MAO-A and MAO-B, in amphistomes. These specific inhibitors also had a differential influence on the in vitro motility of amphistomes, further indicating the involvement of different forms of MAO in the oxidative deamination of biogenic monoamines which might be partly responsible for neuromuscular coordination in amphistomes. The experimental procedures used in this study could be conveniently used for quick screening and evaluation of some of the qualitative effects of anthelmintic drugs under in vitro conditions.

The soluble extracts of Gastrothylax crumenifer isolated from the rumen of buffalo (Bubalus bubalis) were fractionated on a Sephadex G-200 column. A total of eight major fractions (F1, F2, F3, F4, F5, F6, F7, and F8) were separated from the whole homogenate of G. crumenifer, and each of these fractions was tested for their antigenicity by ELISA against rabbit hyperimmune sera. It was observed that F1, F2, F3 and F4 were highly antigenic, F6 and F7 were moderately antigenic and F5 and F8 were poorly antigenic. The individual fractions analysed after SDS-PAGE and Western blotting indicated that the antigenic fractions of G. crumenifer are of low molecular weight, in the range of <14-50kDa, and predominant antigenic components which were evident in most of the Sephadex profiles were of Mr 15, 18, 19, 23-24 and 28-32kDa.

Aim: To determine risk factors for failure of temporary dialysis catheters, we prospectively studied the outcome of 178 non-tunneled dual lumen catheters placed in 126 consecutive patients requiring treatment of acute renal failure (ARF) or end-stage renal disease (ESRD)., Methods: Internal jugular (IJ) or subclavian (SC) catheters were used in 122 instances and femoral catheters were employed in 56., Results: IJ or SC catheters with tips in the right atrium or superior vena cava (n = 112) failed (defined as a blood flow < 250 ml/min) 17% of the time, compared with a 40% failure rate for catheters with more peripherally located tips (n = 10), p < 0.05, chi2 testing. In a multivariate analysis, use in ESRD and location peripheral to the SVC were risk factors for catheter failure. Use of one of three catheter brands was associated with a lower failure rate. Although mean venous pressures at 200 ml/min blood flow were higher in IJ or SC catheters that failed, the presence of a high venous pressure, number of catheter uses, IJ vs. SC placement, inpatient vs. outpatient status, and fresh venipuncture vs. placement over a guidewire passed through a previous catheter did not predict catheter malfunction. With femoral catheters, the only risk factor for failure was use in ESRD., Conclusion: Of the factors that can be influenced by placement technique, catheter tip location is most important. Whether one catheter brand is superior awaits further confirmation.

Vitamin K deficiency is a common occurrence in the surgical and intensive care unit population, but its incidence in kidney and combined kidney-pancreas allograft recipients has not been described. We report four patients who received cadaveric kidney or combined kidney-pancreas allografts and subsequently developed significant bleeding associated with deficiency of vitamin K. Their coagulopathy promptly resolved with the parenteral administration of vitamin K. Treatment with vitamin K should be considered in kidney or combined kidney-pancreas allograft recipients with a prolonged prothrombin or partial thromboplastin time during the first postoperative week to avoid hemorrhagic complications.

Plasminogen activators are known to be involved in the metastatic spread of breast cancer. In the present study we examined the effects of antiestrogens [Analog II (1,1-dichloro-cis-2,3-diphenyl cyclopropane) (AII), ICI-182,780 (ICI) and tamoxifen (TAM)], on the in vitro release of uPA from estrogen receptor (ER)-positive MCF-7 (MCF) and ER-negative MDA-MB-231 (MDA) human breast cancer cell lines. Using a solid-phase radioassay, uPA activity was found to be higher in the culture medium from MDA cells compared to MCF cells. Aminocaproic acid, a specific plasmin inhibitor, produced a 50-60% reduction in the degradation of labeled substrate, in the solid phase assay, using culture medium from both cell lines, thus indicating that most of the proteolysis observed was due to uPA-mediated plasmin generation from plasminogen. In the absence of plasminogen, the enzyme activity was not detected in either the quantitative assay or by zymography. In MDA cells, uPA release was not altered by any of the antiestrogens used alone or in the presence of estradiol. In contrast, in MCF cells, ICI alone produced maximal inhibition (40%) of enzyme release, while estradiol alone produced a 120% increase in enzyme activity. When co-administered with estradiol, in MCF cultures, each antiestrogen reduced enzyme activity to control levels. Substrate gel zymography revealed that the urokinase-type PA is the predominant form of PA released by both cell lines. Comparison of the activity of all three antiestrogens used in this study indicates that ICI is the most potent inhibitor of enzyme activity in ER-positive MCF cells.

The metastasis of malignant tumor cells to other organs in the body is the major cause of cancer-related patient mortality. Therefore, the inhibition of tumor cell motility is critical in the prevention or control of tumor malignancy. In the present study, the antimetastatic potential of antiestrogens [tamoxifen (TAM); ICI-182,780 (ICI); and Analog II (AII)] on highly invasive, estrogen receptor (ER)-negative MDA-MB-231 (MDA) and non-invasive, ER-positive MCF-7 (MCF) human breast cancer cell lines was investigated using an in vitro wound model. Wounds were created in confluent cell cultures and repopulation of the wound space was evaluated by counting the number of cells that migrated into the wound area and by measuring the maximum distance traveled. In addition, the number of cells that were passively seeded into the wounded area was determined. ICI and AII reduced the number of MCF cells that migrated into the wounded area and reduced the number of viable passively seeded MDA cells. Unlike ICI and AII, TAM appeared to enhance MCF and MDA cell movement. This study indicates that the in vitro wound technique is applicable to the study of breast cancer cell movement in response to antiestrogens and other antimetastatic agents. It also demonstrates that antiestrogens differ in their influence on breast cancer cell migration.

Matrix metalloproteinases (MMPs) play an important role in tumor cell invasion and cancer metastasis. Accordingly, a higher level of these enzymes has been associated with the invasive phenotype. In the present study the effect of the antiestrogens, Analog II (AII), ICI-182,780 (ICI), and tamoxifen (TAM), on the in vitro release of MMPs, particularly gelatinases A and B by the MDA-MB-231 (MDA) and MCF-7 (MCF) human breast cancer cell lines was investigated using a solid-phase radioassay and substrate gel zymography. Quantitatively, the enzyme activity was found to be higher in the incubation medium from estrogen receptor (ER)-negative and more metastatic MDA cells compared to ER-positive and less metastatic MCF cells. Tissue inhibitor of metalloproteinases-1 (TIMP-1) reduced the enzyme activity in media from both MDA (56.36%) and MCF (71.03%) cells. Differential antiestrogen effects on the two cell lines were observed following 4 days of treatment of cells at a concentration of 10(-6)M. The enzyme activity from MDA cells was not influenced by treatment with any of the antiestrogens, whereas, in MCF cells, ICI produced the greatest enzyme inhibition (47.93%), followed by AII (36.51%) and TAM (24.05%). Concurrent treatment of MCF cells with 17-beta-estradiol (10(-9)M) partially reversed the AII- and TAM-induced but did not alter ICI-induced inhibition of enzyme activity. Substrate gel zymography revealed that among the MMPs, the MDA cells released predominantly progelatinase A (72 kDa) along with minor bands of activated forms, 62 kDa and 59 kDa, whereas progelatinase B (92 kDa) was detected predominantly in the medium from MCF cells. Comparison of the overall antiestrogen effect indicates that ICI is the most potent inhibitor of enzyme activity in ER-positive MCF cells and that antiestrogen treatment may limit the metastatic potential of ER-positive breast cancer.

The amphistomes Gigantocotyle explanatum and Gastrothylax crumenifer utilize leucine, alanine, proline and methionine during in vitro incubations. Autoradiography on sections of these flukes reveal a time-dependent differential incorporation of tritium-labelled amino acids in various tissues. The tegument appears to be the primary surface through which amino acids are absorbed. Following absorption, the reappearance of [3H]-leucine and [3H]-alanine on the tegumental surface during late chase periods indicates their possible involvement in tegumental secretion. A combination of diffusion and carrier-mediated uptake, possibly involving gamma-glutamyl transpeptidase, is indicated. The transport loci show differences in carrier-affinity (Kt) and maximum uptake velocities (Vmax) for amino acids under study, which suggest multiple transport molecules. Metabolic studies reveal that aspartate, alanine, ornithine, proline, leucine and methionine undergo transamination through 2-oxoglutarate-linked transaminases, distributed in the cytosolic and mitochondrial fractions of G. explanatum and G. crumenifer. With the exception of alanine transaminase, the enzyme levels in the cytosolic fraction were higher than the mitochondrial fraction of the two amphistomes. Predominantly cytosolic glutamate dehydrogenase which was comparatively higher in G. explanatum, catalyse amination of alpha-ketoglutarate. A high level of cytosolic arginase alone does not indicate a functional urea cycle. A tentative pathway of amino acid metabolism in these amphistomes is proposed.

Analysis of various biochemical components during the development of the miracidium of G. explanatum showed marked changes, particularly in glycogen, protein and DNA levels. Though the total lipids remained more or less unchanged, alterations in the levels of cholesterol, triglycerides, free fatty acids, phospholipids and phospholipid fractions were also recorded. Such changes could be intrinsically programmed for the cellular differentiation and organogenesis in larval amphistomes.

The protein content of fresh homogenates and their corresponding TCA precipitated fractions of 10 different species of helminths was estimated by the methods of Lowry et al. and Spector using the Folin phenol reagent and Coomassie brilliant blue G-250 respectively. The former method gives exaggerated values as compared to the latter method. The parasite phenols, phenolic proteins and catecholamines could be responsible for interference in the Lowry's procedure. The TCA noln-precipitable moieties also give colour only with the Folin phenol assay. The pronounced intra-specific differences in the total protein content of helminths reflect their metabolic variations and adaptations. Habitat does not appear to influence the protein content of parasites, however, the effect of host variation was evident in the pouched amphistome G. crumenifer. It is concluded that the dye binding method gives more consistent results and it can be conveniently applied to crude tissue homogenates of helminths.

Biochemical components, glycogen, protein, nucleic acids and lipid fractions were analysed every month from January to December 1986, in the liver and rumen amphistomes Gigantocotyle explanatum and Gastrothylax crumenifer, respectively. The results reveal a considerable seasonal variation. In the rumen amphistome the components reach their maximum level only once a year, whereas in the liver amphistome, more than one peak is observed in a year. In G. crumenifer the content of glycogen and nucleic acids increases before the onset of egg production while protein and lipids reach their maximum level during the egg production phase. The variations in biochemical components were associated with the reproductive cycles and gonad recrudescence of these parasites.

A 77-year-old woman with post-transfusion purpura failed to respond to two 2.5-litre plasma exchanges with albumin as a replacement fluid. However, intravenous infusion of high-dose human immunoglobulin produced a response within 4 h. It is suggested that plasma exchange and exchange transfusion are effective in this condition mainly because they have allowed large doses of immunoglobulin to be infused in the form of plasma or whole blood.

A patient with Klinefelter's syndrome and diabetes mellitus was diagnosed as having myelodysplasia. Cytogenetic analysis of the peripheral blood and the bone marrow cells confirmed the presence of a constitutional 47,XXY chromosome complement. In addition, complex karyotypic abnormalities were present.

Analysis of the major biochemical components of Taenia hydatigena cysticerci collected from goats and pigs showed marked differences, particularly in glycogen, protein, lipid and DNA levels. Differences were also detected in the levels of cholesterol, triglycerides, free fatty acids and phospholipids. Furthermore, the profile of phospholipid fractions revealed quantitative differences between the two species. It is concluded that the cysticerci of goat and pig origin probably represent two different strains and possibly follow the same pattern of speciation as reported in the related taeniid, Echinococcus granulosus.