Inactivation of Cytomegalovirus in Breast Milk Using Ultraviolet-C Irradiation: Opportunities for a New Treatment Option in Breast Milk Banking.

Pasteurized donor human milk is provided by milk banks to very preterm babies where their maternal supply is insufficient or unavailable. Donor milk is currently processed by Holder pasteurization, producing a microbiologically safe product but significantly reducing immunoprotective components. Ultraviolet-C (UV-C) irradiation at 254 nm is being investigated as an alternative treatment method and has been shown to preserve components such as lactoferrin, lysozyme and secretory IgA considerably better than Holder pasteurization. We describe the inactivation of cytomegalovirus, a virus commonly excreted into breast milk, using UV-C irradiation. Full replication was ablated by various treatment doses. However, evidence of viral immediate early proteins within the cells was never completely eliminated indicating that some viral gene transcription was still occurring. In conclusion, UV-C may be a safe alternative to pasteurisation for the treatment of human donor milk that preserves the bioactivity. However, our data suggests that CMV inactivation will have to be carefully evaluated for each device designed to treat breast milk using UV-C irradiation.

Role of IGF signaling in catch-up growth and accelerated temporal development in zebrafish embryos in response to oxygen availability.

Animals respond to adverse environments by slowing down or arresting growth and development. Upon returning to normal conditions, they often show compensatory acceleration in growth and developmental rate. This phenomenon, known as `catch-up' growth, is widely documented in the animal kingdom. The underlying molecular mechanisms, however, are poorly understood. Using the zebrafish embryo as an experimental model system, we tested the hypothesis that changes in IGF signaling activities play an important role in the accelerated growth and temporal development resulting from re-oxygenation following hypoxia. We show that chronic hypoxia reduced, and re-oxygenation accelerated, embryonic growth and developmental rate. Whereas hypoxia repressed the Igf1 receptor and its downstream Erk1/2 and Akt signaling activities, re-oxygenation restored their activities. Specific inhibition of Igf1 receptor signaling during re-oxygenation by genetic and pharmacological approaches attenuated catch-up growth. Further analysis showed that whereas PI3K-Akt is required in both normal and catch-up growth, Mek1/2-Erk1/2 activation induced by elevated IGF signaling during re-oxygenation is particularly crucial for catch-up growth. These results suggest that the evolutionarily conserved IGF signaling pathway coordinates growth and temporal development in zebrafish embryos in response to oxygen availability.

Idiopathic retroperitoneal fibrosis presenting with recurrent bilateral uveitis.

Patients with recurrent uveitis are often evaluated for the presence of underlying systemic disease. The authors describe a 55-year-old black female who presented with isolated recurrent anterior uveitis. Laboratory evaluations were notable for elevated inflammatory markers. She subsequently developed left lower extremity painless swelling; ultrasound evaluation was negative for deep venous thrombosis. CT scan of her abdomen and pelvis demonstrated multiple amorphous soft tissue densities throughout the small bowel mesentery and retroperitoneum, associated with left-sided hydronephrosis. Histopathology of a retroperitoneal mass demonstrated retroperitoneal fibrosis (RPF). Treatment with systemic corticosteroids led to shrinkage of her mesenteric and retroperitoneal masses, resolution of uveitis and normalisation of inflammatory markers. Albeit rare, RPF should be considered in the diagnostic investigation of patients with recurrent uveitis, especially those with abdominopelvic or lower extremity complaints.

Midterm follow-up of opening-wedge high tibial osteotomy.

BACKGROUND: High tibial osteotomy is a valuable option for patients with varus gonarthrosis. To avoid difficulties with closing-wedge osteotomies, medial opening-wedge high tibial osteotomies have been advocated. HYPOTHESIS: Opening-wedge high tibial osteotomy is a good option in highly active patients with varus gonarthrosis who would like to delay or prevent progression to total knee arthroplasty without activity restrictions. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Twenty consecutive patients with varus gonarthrosis were treated with a medial opening-wedge high tibial osteotomy using the Puddu plate and allograft bone graft for a prospective study (14 men and 6 women; average age, 49.4 years [range, 36-67 years]). Gait analysis was performed preoperatively and at 6 months postoperatively. Preoperative radiographs, subjective ratings, and knee scores (Lysholm and Hospital for Special Surgery [HSS] scores) were obtained. At 2 years postoperatively and at the latest follow-up visit (average, 8.3 years), the subjective ratings and knee scores were repeated. RESULTS: Gait analysis revealed an abnormal weightbearing pattern preoperatively with the vertical ground-reaction force. The postoperative vertical ground-reaction force revealed a normal double peak pattern. The preoperative adduction moment was 29% greater than the 6-month postoperative adduction moment. The preoperative varus averaged 3.6° and was corrected to an average of 7.5° of valgus postoperatively. All patients subjectively rated their preoperative knee as poor. At 2 years postoperatively, most patients (14) rated their knee as good, with 5 excellent and only 1 fair rating. The average preoperative Lysholm and HSS knee scores were 54.2 and 75.9, respectively, compared with the 2-year postoperative averages of 89.1 and 92.7, respectively. At 8 years postoperatively, there was 70% survivorship with 42% of patients rating their knees as good or excellent. Five patients (25%) had undergone total knee arthroplasty. Lysholm and HSS knee scores were 83.0 and 86.8, respectively, for the surviving knees at 8 years postoperatively. CONCLUSION: Medial opening-wedge high tibial osteotomy produces good results in the midterm. After the osteotomy, a more normal appearing weightbearing pattern with double peaks was seen. The adduction moment significantly decreased, resulting in less contact pressure through the medial degenerative compartment of the knee. The authors recommend medial opening-wedge high tibial osteotomy for young patients with varus alignment and medial compartment arthritis to allow this patient population to remain highly active and delay progression to total knee arthroplasty without activity restrictions.

Antiparasitic activities of novel, orally available fumagillin analogs.

Fumagillin, an irreversible inhibitor of MetAP2, has been shown to potently inhibit growth of malaria parasites in vitro. Here, we demonstrate activity of fumagillin analogs with an improved pharmacokinetic profile against malaria parasites, trypanosomes, and amoebas. A subset of the compounds showed efficacy in a murine malaria model. The observed SAR forms a basis for further optimization of fumagillin based inhibitors against parasitic targets by inhibition of MetAP2.

Evaluation of cryptolepine and huperzine derivatives as lead compounds towards new agents for the treatment of human African trypanosomiasis.

The alkaloid cryptolepine (1) and eight synthetic analogues (2-8) were assessed for in vitro activities against Trypanosoma brucei. Four of the analogues were found to be highly potent with IC50 values of less than 3 nM and three of these were assessed against T. brucei brucei infection in rats. The most effective compound was 2, 7-dibromocryptolepine (7); a single oral dose of 20 mg/kg suppressed parasitaemia and increased the mean survival time to 13.6 days compared with 8.4 days for untreated controls. In addition, four huperzine derivatives (9-12) were shown to have in vitro antitrypanosomal activities with IC50 values ranging from 303-377 nM.

The many faces of amyloid beta in Alzheimer's disease.

The 'amyloid cascade hypothesis' links amyloid beta peptide (Abeta) with the pathological process of Alzheimer's disease (AD) and it still awaits universal acceptance. Amyloid precursor protein (APP), through the actions of the gamma-secretase complex, eventually becomes a different Abetaspecies. The various Abeta species have proven to be difficult to investigate under physiological conditions, and the species of Abeta responsible for neurotoxicity has yet to be unequivocally identified. The two important Abeta peptides involved are Abeta(1-40) and Abeta(1-42), and each has been ascribed both toxic and beneficial attributes. The ratio between the two species can be important in AD etiology. Additionally, shorter variants of Abeta peptides such as Abeta(1-8), Abeta(9-16) and Abeta(16) have also been shown to be potential participants in AD pathology. Interestingly, a new 56-kDa Abeta peptide (Abeta*56) disrupts memory when injected into the brains of young rats. Transgenic mice models are complicated by the interplay between various human Abeta types and the mouse Abeta types in the mouse brains. However, the accumulation of Abeta(1-42) in the brains of transgenic C. elegans worms and Drosophila is indeed detrimental. A less investigated aspect of AD is epigenetics, but in time the investigation of the role of epigenetics in AD may add to our understanding of the development of AD.

Synthesis and anti-breast cancer activities of substituted quinolines.

Promising anti-breast cancer agents derived from substituted quinolines were discovered. The quinolines were readily synthesized in a large scale from a sequence of reactions starting from 4-acetamidoanisole. The Michael addition product was isolated as the reaction intermediate in the ring closing reaction of 4-amino-5-nitro-2-(3-trifluoromethylphenyloxy)anisole with methyl vinyl ketone leading to 6-methoxy-4-methyl-8-nitro-5-(3-trifluoromethylphenyloxy)quinoline (14). The amino function of 8-amino-6-methoxy-4-methyl-5-(3-trifluoromethylphenyloxy)quinoline, prepared from 14, was connected to various side chains via alkylation with N-(3-iodopropyl)phthalimide, Michael addition with acrylonitrile, and reductive amination with various heterocycle carboxaldehydes, such as imidazole-4-carboxaldehyde, thiophene-2-carboxaldehyde, and 2-furaldehyde. Effects of the substituted quinolines on cell viability of T47D breast cancer cells using trypan blue exclusion assay were examined. The results showed that the IC(50) value of 6-methoxy-8-[(2-furanylmethyl)amino]-4-methyl-5-(3-trifluoromethylphenyloxy)quinoline is 16+/-3nM, the lowest IC(50) out of all the quinolines tested. IC(50) values of three other quinolines are in the nanomolar range, a desirable range for pharmacological testing.

Total syntheses of (+/-)-ovalicin, C4(S *)-isomer, and its C5-analogs and anti-trypanosomal activities.

Total syntheses of (+/-)-ovalicin, its C4(S( *))-isomer 44, and C5-side chain intermediate 46 were accomplished via an intramolecular Heck reaction of (Z)-3-(tert-butyldimethylsilyloxy)-1-iodo-1,6-heptadiene and a catalytic amount of palladium acetate. Subsequent epoxidation, dihydroxylation, methylation, and oxidation led to (3S( *),5R( *),6R( *))-5-methoxy-6-(tert-butyldimethylsilyloxy)-1-oxaspiro[2.5]octan-4-one (2), a reported intermediate. The addition of a side chain with cis-1-lithio-1,5-dimethyl-1,4-hexadiene (27) followed by oxidation afforded (+/-)-ovalicin. The functional group manipulation afforded a number of regio- and stereoisomers, which allow the synthesis of analogs for bioevaluation. The structure of 44 was firmly established via a single-crystal X-ray analysis. The stereochemistry at C4 generated from the addition reactions of alkenyllithium with ketones 2, 40, and 45 is dictated by C6-alkoxy functionality. Anti-trypanosomal activities of various ovalicin analogs and synthetic intermediates were evaluated, and C5-side chain analog, 46, shows the strongest activity. Compound 44 shows antiproliferative effect against HL-60 tumor cells in vitro. Compounds 46 and a precursor, (3S( *),4R( *),5R( *),6R( *))-5-methoxy-4-[(E)-(1',5'-dimethylhexa-1',4'-dienyl)]-6-(tert-butyldimethylsilyloxy)-1-oxaspiro[2.5]octan-4-ol (28), may be explored for the development of anti-parasitic drugs.

Discovery of trypanocidal compounds by whole cell HTS of Trypanosoma brucei.

Chemotherapy against human African trypanosomiasis relies on four drugs that cause frequent and occasionally severe side-effects. Because human African trypanosomiasis is a disease of poor people in Africa, the traditional market-driven pathways to drug development are not available. One potentially rapid and cost-effective approach to identifying and developing new trypanocidal drugs would be high throughput-screening of existing drugs already approved for other uses, as well as clinical candidates in late development. We have developed an ATP-bioluminescence assay that could be used to rapidly and efficiently screen compound libraries against trypanosomes in a high throughput-screening format to validate this notion. We screened a collection of 2160 FDA-approved drugs, bioactive compounds and natural products to identify hits that were cytotoxic to cultured Trypanosoma brucei at a concentration of 1 mum or less. This meant that any hit identified would be effective at a concentration readily achievable by standard drug dosing in humans. From the screen, 35 hits from seven different drug categories were identified. These included the two approved trypanocidal drugs, suramin and pentamidine, several other drugs suspected but never validated as trypanocidal, and 17 novel trypanocidal drugs.

Malaria: therapy, genes and vaccines.

Malaria kills over 3,000 children each day. Modern molecular and biochemical approaches are being used to help understand and control Plasmodium falciparum, the parasite that causes this deadly disease. New drugs are being invented for both chemoprophylaxis and therapeutic treatments and their use is discussed along side that of the more commonly used treatments. Classical genetic crosses coupled with molecular analysis of gene loci are use to explain the genetics behind the development of specific drug resistances that the parasites have naturally developed. Rapid advances in DNA sequencing techniques have allowed the compete sequencing of the P. falciparum and several other rodent malaria parasite genomes. Proteomics and computational analysis of these vast databanks are being used to model and investigate the three-dimensional structure of many key malaria proteins in an attempt to facilitate drug design. Recombinant protein expression in bacteria and yeast coupled with cGMP purification technologies and conditions have lead to the recent availability of several dozen malaria protein antigens for human-use Phase I and Phase II vaccine trials. Drug companies, private foundations, and key government agencies have contributed to the coordinated efforts needed to test these antigens, adjuvants and delivery methods in an effort to find an effective malaria vaccine that will prevent infection and disease.

Antitrypanosomal activity of 5'-deoxy-5'-(iodomethylene)adenosine and related 6-N-cyclopropyladenosine analogues.

Treatment of the 6-N-cyclopropyl-2',3'-di-O-isopropylideneadenosine 5'-aldehyde with sulfone-stabilized phosphonate or fluorophosphonate reagents followed by stannyldesulfonylations and subsequent iodo- or protiodestannylation gave 6-N-cyclopropyl-5'-deoxy-5'-(iodomethylene)adenosine 8b or its 5'-fluoromethylene analogue 11. Treatment of the 5'-aldehyde with hydroxylamine or dibromomethylene- or cyanomethylene-stabilized Wittig reagents and deprotections gave the oxime 4b, 5'-cyanomethylene 5b, and 5'-dibromomethylene 13b analogues. Dehydrobromination of 13b gave acetylenic compound 14b. From the tested 6-N-cyclopropyladenosine analogues modified at the 5' carbon, the 5'-iodomethylene 8b had the most potent activity against Trypanosoma brucei in vitro with an IC50 of 12 microg/mL. The IC50 value was 19 microg/mL for both the 5'-fluoromethylene 11 and the 5'-cyanomethylene 5b compounds. The (E)-5'-deoxy-5'-(iodomethylene)adenosine 2a, a known inhibitor of AdoHcy hydrolase not modified with a cyclopropyl ring at 6-amino group, also inhibited T. brucei with an IC50 of 9 microg/mL. In contrast to some other adenosine analogues modified at C5', the 6-N-cyclopropyladenosine analogues described here do not exhibit an inhibitory effect on AdoHcy hydrolase and displayed only marginal antiviral activity.

Isolated acetabular revision through the posterior approach: short-term results after revision of a recalled acetabular component.

Isolated acetabular revision is commonly associated with high rates of postoperative dislocation. We hypothesize that the dislocation rate in a series of isolated acetabular revisions for loosening of recalled acetabular components is low when strict intraoperative stability testing is coupled with re-repair of the piriformis tendon. Twenty-six isolated revisions of aseptic, loose recalled acetabular components were performed through the posterior approach by a single surgeon. All piriformis tendon and posterior capsular repairs from the index operation were considered intact at the time of revision. The piriformis tendon was re-repaired in each revision case. The average revision acetabular component was 3 mm larger than the index component. In this select group of isolated acetabular revisions, strict intraoperative stability testing and re-repair of the piriformis tendon yielded zero dislocations.

Osteolysis of the pelvis: evaluation and treatment.

Osteolysis of the pelvis is a common and well-recognized complication associated with total hip arthroplasty. The diagnosis and treatment of osteolysis of the pelvis is a challenging and controversial problem. Osteolysis of the pelvis often is asymptomatic and does not present with symptoms until considerable bone loss and loosening of the acetabular socket occur. Radiographs are the most common way to detect and monitor osteolysis around an implant. However, lesions viewed radiographically usually are underestimations of the lesions found intraoperatively. Moreover, some advocate computed tomography scanning to evaluate these lesions. The indications for treatment of osteolysis with cemented acetabular components are defined more clearly than with a cementless component. If the cemented or cementless acetabular component is loose, then revision is necessary. However, it is less clear when to intervene surgically with a well-fixed cup with osteolysis. Many early reports advocated the removal of a well-fixed socket during revision surgery for osteolysis and polyethylene wear. However, the removal of a well-fixed socket has the potential for significant damage and loss of the surrounding bone resulting in loss of integrity of a column or pelvic discontinuity, which may compromise placing another acetabular component. Recently, a new treatment strategy of retaining a well-fixed socket, exchanging the liner, and grafting lesions has proven successful. Without the removal of the acetabular shell, different techniques are needed to graft the osteolytic lesions. Osteolysis is a difficult problem; however, with radiographic surveillance to monitor patients for lesions, proper indications, and good surgical techniques, the treatment of osteolysis of the pelvis can result in a well-functioning total hip arthroplasty.

Antiprotozoal activities of symmetrical bishydroxamic acids.

Symmetrical bishydroxamic acids along with their sodium salts containing an alkyl spacer between two aromatic rings were synthesized, and their antiparasitic activities were evaluated. Bishydroxamic acids were conveniently prepared from the alkylation of methyl 4-hydroxybenzoate with various dihalo-alkane, -alkene, and -ether followed by reaction with hydroxylamine. Surprisingly, the bishydroxamic acids and their sodium salts possess strong inhibitory activities against Plasmodium falciparum parasites with IC50 values in the range of 0.26-3.2 microM. Bishydroxamic acid 3 and its sodium salt 12 also inhibit the growth of Leishmania donovani, albeit at higher concentrations. The corresponding biscarboxylic acids and bismethyl esters are inactive. Presumably, the ability of bishydroxamic acids to complex with metallic iron in hemoglobin may be responsible for antimalarial activity of these compounds.

Anti-HIV-1 activity of 3-deaza-adenosine analogs. Inhibition of S-adenosylhomocysteine hydrolase and nucleotide congeners.

Eight adenosine analogs, 3-deaza-adenosine (DZA), 3-deaza-(+/-)aristeromycin (DZAri), 2',3'-dideoxy-adenosine (ddAdo), 2',3'-dideoxy-3-deaza-adenosine (ddDZA), 2',3'-dideoxy-3-deaza-(+/-)aristeromycin (ddDZAri), 3-deaza-5'-(+/-)noraristeromycin (DZNAri), 3-deaza-neplanocin A (DZNep), and neplanocin A (NepA), were tested as inhibitors of human placenta S-adenosylhomocysteine (AdoHcy) hydrolase. The order of potency for the inhibition of human placental AdoHcy hydrolase was: DZNep approximately NepA >> DZAri approximately DZNAri > DZA >> ddAdo approximately ddDZA approximately ddDZAri. These same analogs were examined for their anti-HIV-1 activities measured by the reduction in p24 antigen produced by 3'-azido-3'-deoxythymidine (AZT)-sensitive HIV-1 isolates, A012 and A018, in phytohemagglutinin-stimulated peripheral blood mononuclear (PBMCs) cells. Interestingly, DZNAri and the 2',3'-dideoxy 3-deaza-nucleosides (ddAdo, ddDZAri, and ddDZA) were only marginal inhibitors of p24 antigen production in HIV-1 infected PBMC. DZNAri is unique because it is the only DZA analog with a deleted methylene group that precludes anabolic phosphorylation. In contrast, the other analogs were potent inhibitors of p24 antigen production by both HIV-1 isolates. Thus it was postulated that these nucleoside analogs could exert their antiviral effect via a combination of anabolically generated nucleotides (with the exception of DZNAri), which could inhibit reverse transcriptase or other viral enzymes, and the inhibition of viral or cellular methylation reactions. Additionally, QSAR-like models based on the molecular mechanics (MM) were developed to predict the order of potency of eight adenosine analogs for the inhibition of human AdoHcy hydrolase. In view of the potent antiviral activities of the DZA analogs, this approach provides a promising tool for designing and screening of more potent AdoHcy hydrolase inhibitors and antiviral agents.

Structure, evolution, and inhibitor interaction of S-adenosyl-L-homocysteine hydrolase from Plasmodium falciparum.

S-adenosylhomocysteine hydrolase (SAHH) is a key regulator of S-adenosylmethionine-dependent methylation reactions and an interesting pharmacologic target. We cloned the SAHH gene from Plasmodium falciparum (PfSAHH), with an amino acid sequence agreeing with that of the PlasmoDB genomic database. Even though the expressed recombinant enzyme, PfSAHH, could use 3-deaza-adenosine (DZA) as an alternative substrate in contrast to the human SAHH, it has a unique inability to substitute 3-deaza-(+/-)aristeromycin (DZAri) for adenosine. Among the analogs of DZA, including neplanocin A, DZAri was the most potent inhibitor of the PfSAHH enzyme activity, with a K(i) of about 150 nM, whether Ado or DZA was used as a substrate. When the same DZA analogs were tested for their antimalarial activity, they also inhibited the in vitro growth of P. falciparum parasites potently. Homology-modeling analysis revealed that a single substitution (Thr60-Cys59) between the human and malarial PfSAHH, in an otherwise similar SAH-binding pocket, might account for the differential interactions with the nucleoside analogs. This subtle difference in the active site may be exploited in the development of novel drugs that selectively inhibit PfSAHH. We performed a comprehensive phylogenetic analysis of the SAHH superfamily and inferred that SAHH evolved in the common ancestor of Archaea and Eukaryota, and was subsequently horizontally transferred to Bacteria. Additionally, an analysis of the unusual and uncharacterized AHCYL1 family of the SAHH paralogs extant only in animals reveals striking divergence of its SAH-binding pocket and the loss of key conserved residues, thus suggesting an evolution of novel function(s).

Gene expressions in Jurkat cells poisoned by a sulphur mustard vesicant and the induction of apoptosis.

1. The sulphur mustard vesicant 2-chloroethylethyl sulphide (CEES) induced apoptosis in Jurkat cells. 2. Akt (PKB), a pivotal protein kinase which can block apoptosis and promotes cell survival, was identified to be chiefly down-regulated in a dose-dependent manner following CEES treatment. Functional analysis showed that the attendant Akt activity was simultaneously reduced. 3. PDK1, an upstream effector of Akt, was also down-regulated following CEES exposure, but two other upstream effectors of Akt, PI3-K and PDK2, remained unchanged. 4. The phosphorylation of Akt at Ser(473) and Thr(308) was significantly decreased following CEES treatment, reflecting the suppressed kinase activity of both PDK1 and PDK2. 5. Concurrently, the anti-apoptotic genes, Bcl family, were down-regulated, in sharp contrast to the striking up-regulation of some death executioner genes, caspase 3, 6, and 8. 6. Based on these findings, a model of CEES-induced apoptosis was established. These results suggest that CEES attacked the Akt pathway, directly or indirectly, by inhibiting Akt transcription, translation, and post-translation modification. 7. Taken together, upon exposure to CEES, apoptosis was induced in Jurkat cells via the down-regulation of the survival factors that normally prevent the activation of the death executioner genes, the caspases.

Failure of a retinacular flap to prevent dorsal wrist pain after titanium Pi plate fixation of distal radius fractures.

Patients with distal radius fractures that had been treated with a dorsal Pi plate and retinacular flap covering the transverse limb of the Pi plate were evaluated clinically and radiographically. Nine of 20 patients (45%) required plate removal for dorsal wrist pain. Three of the remaining 11 who retained the plate had dorsal tenderness over the wrist extensors. There were no differences evident between the 2 groups in plate size, position, or number of screws used. In addition there were no significant differences between the groups in either radial height or inclination. The palmar tilt did show a trend toward statistical significance: those patients who required plate removal had an average of 4.1 degrees of dorsal tilt, patients whose plate was not removed averaged 2.8 degrees of palmar tilt. Our results show that the retinacular flap covering the distal transverse limb of the Pi plate did not prevent the occurrence of dorsal wrist pain. Dorsal wrist pain remained a problem with dorsal plating of distal radius fractures.