STUDIES ON NORMAL AND IMMUNE LYMPHOCYTE TRANSFER REACTIONS IN GUINEA PIGS, WITH SPECIAL REFERENCE TO THE CELLULAR CONTRIBUTION OF THE HOST

Using guinea pigs of strains 2 and 13 and their F₁ hybrids as experimental subjects, various lines of evidence have been obtained that in this species, as in all others tested, the only significant cellular antigens with which donor lymphocytes engage when normal and immune lymphocyte reactions are incited are radiosensitive leukocytes. Constitutive cells of the skin are unimportant. (a) The intensities of these reactions in irradiated subjects are dependent upon the peripheral leukocyte concentration. When this falls below a certain threshold no reactions are incitable. (b) Highly leukopenic animals are capable of developing immune lymphocyte transfer (ILT) reactions if normal lymphoid cells of their own genetic constitution are mixed with the putative attacking donor cells, as "supplementing antigen," before inoculation. (c) Radiation-chimeric strain 13 animals having F₁ hybrid leukocytes in their bloodstream give typical ILT reactions when challenged intradermally with strain 13 anti-2 node cells. Exposure of strain 2 animals to 600 R does not prevent their becoming actively immunized if, 24 hr later, they are injected intradermally with strain 13 lymphocytes. However, this sensitization, revealed by the host's capacity to give delayed hypersensitivity reactions, wanes as leukopenia progresses. On the basis of this and other findings it is argued that the flare-up stage of the NLT reaction in preirradiated hosts is mainly an expression of host sensitivity against the transferred alien cells. Two unexpected observations have been made in the course of this study: (a) F₁ hybrid animals developed what appeared to be a strong delayed hypersensitivity after intradermal inoculation with parental strain lymphoid cells or antigenic extracts prepared from them. (b) If strain 13 guinea pigs which had been sensitized against strain 2 tissue antigens by intradermal injection of lymphocytes 7 days beforehand were inoculated intravenously with strain 2 antigenic extract a significant proportion of the animals developed severe delayed necrotizing reactions, recall flares, at some or all of the healed skin inoculation sites.     

The contribution of peroxisomes to lipid metabolism

Peroxisomes are ubiquitous subcellular organelles. They contain catalase and hydrogen peroxide-producing oxidases like fatty acyl-CoA oxidase. The latter enzyme is part of a special fatty acid beta-oxidation system which shortens long-chain fatty acids. The middle-chain acids formed are subsequently degraded by mitochondria. The capacity to remove very long fatty acids and trans-unsaturated acids found in hydrogenated oils is restricted to peroxisomes. Essentially, the peroxisomal beta-oxidation system is not constitutive but inducible by certain hypolipidaemic compounds which are distinguished by their capacity to lead to proliferation of peroxisomes. Thyroid hormones as well as prolonged exposure to cold and high fat diets, esp. with long-chain unsaturated fatty acids, also induce beta-oxidation and peroxisome proliferation. Two other beta-oxidative reactions namely the removal of the cholesterol side-chain, leading to the formation of bile acids, and the degradation of dicarboxylic acids as formed by omega-oxidation of fatty acids were shown to be connected with peroxisomes. Presumably also 3-hydroxy-3-methyl-glutaryl-CoA reductase, the key enzyme of cholesterol biosynthesis exists in a peroxisomal moiety. NADPH consumed in this reaction (and in the dihydroxyacetone phosphate pathway of glycerolipid synthesis) might be provided by glucose-6-phosphate dehydrogenase which was recently also found in peroxisomes. Peroxisomes are indispensable in forming saturated ether lipids and plasmalogens because alkyldihydroxyacetone phosphate synthase is a membrane enzyme exclusively located in peroxisomes. Certain other enzymes of the dihydroxyacetone phosphate pathway of glycerolipid synthesis are also found in peroxisomes. Because of the combination of oxidases like fatty acyl-CoA oxidase and catalase and the feasibility of reoxidising NADH within the peroxisomes the aerobic metabolism of peroxisomes is energy-wasting. Therefore they might be important in chemical thermogenesis and in the control of body weight. For all these reasons peroxisomes must be essential for human metabolism. This is further demonstrated by genetically caused disorders: Total absence of peroxisomes is connected with the fatal cerebro-hepatorenal Zellweger syndrome. Defective peroxisomal beta-oxidation is manifested in Schilder's disease (adrenoleukodystrophy) characterized by accumulation of very long fatty acids. Peroxisomes perform a number of complementary and auxiliary reactions in general cell metabolism, in particular the cata- and anabolism of certain lipids, and therefore deserve consideration in clinical chemistry.     

Isolation and characterization of novel human immunodeficiency virus integrase inhibitors from fungal metabolites.

We have identified a series of novel inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase by randomly screening natural product extracts using an in vitro biochemical assay designed to identify inhibitors of integrase-catalysed strand transfer. Equisetin recovered from the fungus Fusarium heterosporum and a novel enantiomeric homologue of equisetin from Phoma sp. were isolated as inhibitors of HIV-1 integrase in vitro. Two additional analogues, a novel decalin derivative, integric acid, and oteromycin were also discovered to be inhibitors of integrase. Equisetin and related compounds inhibit 3' end-processing and strand transfer as well as disintegration catalysed by either the full-length enzyme or the truncated integrase core domain (amino acids 50-212). These compounds also inhibit strand transfer reactions catalysed by stable complexes assembled in vitro and integration reactions catalysed by pre-integration complexes isolated from HIV-1-infected cells. The compounds described in this report are structurally novel and mechanistically distinct from many previously described inhibitors of HIV-1 integrase. These results demonstrate the utility of using an appropriately configured assay to identify compounds that are effective post-assembly and the potential of isolating novel integrase inhibitors from complex natural product extracts.     

New regimens for intravenous acetylcysteine,where are we now?

Acetylcysteine has been used as a treatment for paracetamol overdose as a 20.25- or 21-h infusion for nearly 40 years. These regimens give 50% of the dose in the first 15 min or 1 h, and are associated with high rates of adverse reactions. A randomised controlled trial has demonstrated that a shorter (12 h) and simpler (two infusions) acetylcysteine regimen using a slower initial infusion rate produces lower rates of adverse events than the original 20.25-h regimen. However, this study was not sufficiently large to show therapeutic equivalence as a hepatoprotective therapy in paracetamol overdose. Two further studies are now reported, which also suggest lower rates of adverse reactions with lower initial rates of acetylcysteine administration. These modified regimens can now be accepted as better tolerated, but it is unlikely that a randomised study of sufficient size to demonstrate non-inferiority of any novel regimen would ever be funded. Against this background we suggest what can be done to establish the efficacy of these less toxic and potentially shorter alternative acetylcysteine regimens and to establish them into routine clinical use.     

Approach to nigericin derivatives and their therapeutic potential

A new nigericin analogue that has been chemically modified was synthesized through a fluorination process from the parent nigericin, produced from a novel Streptomyces strain DASNCL-29. Fermentation strategies were designed for the optimised production of nigericin molecule and subjected for purification and structural analysis. The fermentation process resulted in the highest yield of nigericin (33% (w/w)). Initially, nigericin produced from the strain DASNCL-29 demonstrated polymorphism in its crystal structure, i.e., monoclinic and orthorhombic crystal lattices when crystallised with methanol and hexane, respectively. Furthermore, nigericin produced has been subjected to chemical modification by fluorination to enhance its efficacy. Two fluorinated analogues revealed that they possess a very potent antibacterial activity against Gram positive and Gram negative bacteria. To date, the nigericin molecule has not been reported for any reaction against Gram-negative bacteria, which are increasingly becoming resistant to antibiotics. For the first time, fluorinated analogues of nigericin have shown promising activity. In vitro cytotoxicity analysis of fluorinated analogues demonstrated tenfold lesser toxicity than the parent nigericin. This is the first type of study where the fluorinated analogues of nigericin showed very encouraging activity against Gram-negative organisms; moreover, they can be used as a candidate for treating many serious infections.

A new nigericin analogue that has been chemically modified was synthesized through a fluorination process from the parent nigericin, produced from a novel Streptomyces strain DASNCL-29.     

New insulins and insulin therapy

The introduction of the so-called 'designer' insulins, the insulin analogues, has offered new opportunities in the clinical management of diabetes. Two additional new entities are close to reaching clinical practice. Linjeta? (formally called VIAject) is not an analogue but rather a different formulation of human insulin which may give it a more rapid onset of action, potentially even faster than the currently available rapid-acting insulin analogues. Degludec?, on the other hand, is an insulin analogue molecule with an ultra-long clinical profile derived from the soluble multi-hexamer formation, resulting in a continuous slow and stable release of insulin degludec monomers which may last longer than currently available long-acting analogues. As with any new type of drug, the safety of the 'designer' insulins has to be closely scrutinised. Last year the increased cancer risk in diabetes entered the spotlight and the potential role of insulin analogues led to controversial discussions. In spite of recent new in vitro and observational data no new conclusive evidence became available. The need for multiple well-conducted and appropriately designed prospective observational studies to follow up the effectiveness and safety of the new insulins and the new insulin treatment regimens remains. In this chapter it was our mission to chose articles published about "new insulins" over the last year that have the most important contribution for the on-going development of ultra-fast- and ultra-long-acting insulin analogs and preparations and their potential side-effects, particularly cancer. This has been done by means of PubMed searches as well as a review of abstracts of the recent large international diabetes meetings such as ADA, EASD and ISPAD.     

Reviewing a plethora of oxidative-type reactions catalyzed by whole cells of Streptomyces species

Selective oxidation reactions represent a challenging task for conventional organic chemistry. Whole-cell biocatalysis provides a very convenient, easy to apply method to carry out different selective oxidation reactions including chemo-, regio-, and enantio–selective reactions. Streptomyces species are important biocatalysts as they can catalyze these selective reactions very efficiently owing to the wide diversity of enzymes and enzymatic cascades in their cell niche. In this review, we present and analyze most of the examples reported to date of oxidative reactions catalyzed by Streptomyces species as whole-cell biocatalysts. We discuss 33 different Streptomyces species and strains and the role they play in different oxidative reactions over the past five decades. The oxidative reactions have been classified into seven categories that include: hydroxylation of steroids/non-steroids, asymmetric sulfoxidations, oxidation of aldehydes, multi-step oxidations, oxidative cleavage, and N-oxidations. The role played by Streptomyces species as recombinant hosts catalyzing bio-oxidations has also been highlighted.

Types of oxidation reactions catalyzed by whole cells of different Streptomyces species.     

Sulfur,mercury, and boron adducts of sydnone imine derived anionic N-heterocyclic carbenes

The sydnone imines (5-benzoylimino)-3-(2-methoxyphenyl)-sydnone imine and molsidomine were deprotonated at C4 to give sydnone imine anions which can be represented as anionic N-heterocyclic carbenes, respectively. Trapping reactions with sulfur gave unstable sydnone imine sulfides which were stabilized by the formation of methyl thioethers, methyl sulfoxides, gold complexes [(PPh₃)Au–S-sydnone imine] and a bis(ligand) mercury(ii) complex. The latter possesses a tetrahedral coordination of the mercury central atom to the sulfur atoms with the N6 nitrogen atoms coordinating as neutral ligands. Water converted the molsidomine anion into ethyl(2-morpholino-2-thioxoacetyl)carbamate. Mercury(ii)chloride and triphenylborane were employed to trap the sydnone imine carbenes as mercury complexes as well as BPh₃ adducts.

The sydnone imines (5-benzoylimino)-3-(2-methoxyphenyl)-sydnone imine and molsidomine were deprotonated at C4 to give sydnone imine anions which can be represented as anionic N-heterocyclic carbenes, respectively.     

Soluble asphaltene oxide: a homogeneous carbocatalyst that promotes synthetic transformations

Carbocatalysts, materials which are predominantly composed of carbon and catalyze the synthesis of organic or inorganic compounds, are promising alternatives to metal-based analogues. Even though current carbocatalysts have been successfully employed in a broad range of synthetic transformations, they suffer from a number of drawbacks in part due to their heterogeneous nature. For example, the insolubility of prototypical carbocatalysts, such as graphene oxide (GO), may restrict access to catalytically-active sites in a manner that limits performance and/or challenges optimization. Herein we describe the preparation and utilization of soluble asphaltene oxide (sAO), which is a novel material that is composed of oxidized polycyclic aromatic hydrocarbons and is soluble in a wide range of organic solvents as well as in aqueous media. sAO promotes an array of synthetically useful transformations, including esterifications, cyclizations, multicomponent reactions, and cationic polymerizations. In many cases, sAO was found to exhibit higher catalytic activities than its heterogeneous analogues and was repeatedly and conveniently recycled, features that were attributed to its ability to form homogeneous phases.

Soluble carbocatalysts, materials which are predominantly composed of carbon and catalyze the synthesis of organic or inorganic compounds, are promising alternatives to metal-based analogues.     

Prognostic value of the reactions of resuscitated neurological patients during hyperbaric oxygenation

A complex of various physiological reactions encountered in critically ill patients with brain vascular disturbances and brain traumas in the course of hyperbaric oxygenation (NBO) have been studied. Different nature of reactions has been established in patients who survived and in those with lethal outcome. Two main types of reactions have been identified: prognostically favourable and prognostically unfavourable. The difference is associated with the oxygen reactivity of the affected brain and body. The reactivity was either retained or damaged, which made NBO effective in some cases and ineffective in others.     

Structure-activity relationships for a series of peptidomimetic antimicrobial prodrugs containing glutamine analogues

Synthetic glutamine analogues such as N3-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid (FMDP) inhibit purified glucosamine-6-phosphate synthase, an intracellular enzyme that is essential for microbial cell wall synthesis, but they are inactive against intact organisms because they cannot enter the cell. However, when the analogues are linked to a peptide they can be actively transported, and FMDP peptidomimetics show broad-spectrum antimicrobial activity. To characterize this process in more detail, the antibacterial activities of various synthetic peptidomimetics containing glutamine analogues have been determined against isogenic strains of Escherichia coli in which one or more of its three peptide transporters Dpp, Opp and Tpp have been mutated. In addition, their affinities for DppA and OppA, the binding-protein components of the transporters, have been measured. In general, antibacterial activities against the various transport mutants correlated with binding to DppA and OppA. Xaa-FMDP compounds have greater activities than FMDP-Xaa analogues. To explore structure-activity relationships for the peptidomimetics, molecular modelling was used to determine the conformational forms they adopt in solution. The relative bioactivities of the peptidomimetics correlated with the percentage of conformers that had backbone torsions matching those previously defined for the molecular recognition templates of the peptide transporters. However, the large size of the N-terminal residue in the FMDP-Xaa analogues appears to interfere with transport and thus to limit antibacterial activity. Overall, the results provide the structural rationale for the identification in silico of analogues with optimal bioactivities, which decreases the need for extensive chemical syntheses and testing.     

Hemolysis during percutaneous mechanical thrombectomy can mimic a hemolytic transfusion reaction

BACKGROUND: Interventional radiologists have developed percutaneous mechanical thrombectomy (PMT) devices to remove intravascular thrombi. Hemolysis, secondary to thrombus destruction from these devices, has been described in radiology journals, but similar reports appear to be lacking in the transfusion medicine literature. Two cases of hemolysis after PMT are described that involved the transfusion service, one of which was reported as a hemolytic transfusion reaction. CASE REPORTS: The first patient received 4 units of red cells (RBCs) during a thrombectomy and subsequent placement of a transjugular intrahepatic portosystemic shunt. The patient developed hemoglobinuria, and it was reported to the blood bank as a possible hemolytic transfusion reaction. After RBC mismatch and bacterial contamination were excluded, the hemolysis was attributed to thrombectomy-related mechanical hemolysis. In the second case, a hemolyzed sample was sent to the blood bank for a type and cross-match. Upon requesting that the sample be redrawn, it was learned that the sample was obtained after PMT. CONCLUSION: Patients who have undergone PMT can have clinical and laboratory findings suggestive of hemolytic transfusion reactions. Although interventional radiologists are familiar with these side effects, the blood bank profession needs to be aware that these procedures cause nonimmune hemolysis and must consider this possibility when evaluating transfusion reactions in these patients.     

STUDIES ON THE OXIDATION-REDUCTION OF HEMOGLOBIN AND METHEMOGLOBIN : II. THE OXIDATION OF HEMOGLOBIN AND REDUCTION OF METHEMOGLOBIN BY ANAEROBIC BACILLI AND BY STERILE PLANT TISSUE.

The oxidation-reduction systems of anaerobic bacteria manifest their action upon the blood pigments by marked deoxygenation of oxyhemoglobin, prompt reduction of methemoglobin to hemoglobin, and to a less marked degree oxidation of hemoglobin to methemoglobin. The action of sterile plant tissue upon oxyhemoglobin, hemoglobin, and methemoglobin has been studied. The oxidations and reductions of the blood pigments which are induced by sterile plant tissue are similar to the reactions which are brought about by pneumococci and by anaerobic bacteria.     

METHODS FOR THE DETERMINATION OF PNEUMOCOCCUS TYPES

The determination of pneumococcus types in lobar pneumonia is of value in the field of prognosis and as a prerequisite for specific serum therapy. The method for the determination of types should be as rapid as possible and a standard technique should be employed. The most satisfactory method is by the intraperitoneal inoculation of a mouse with the patient''s sputum, by which means a rapid and abundant growth of the pneumococcus is obtained and secondary organisms are rapidly eliminated in most instances. The diagnosis of type is made directly on the peritoneal exudate. Certain factors in the method commonly used have interfered with the rapid determination of types in an appreciable number of cases, notably the growth of other organisms in the peritoneal exudate together with the pneumococcus, and some confusion has arisen because occasional strains of pneumococci have been encountered that show cross agglutination reactions when undiluted immune serum is used. Such reactions have been shown to be due to a limited zone of non-specific immunity and they in no way invalidate the classification of the pneumococci into sharply defined immunological groups. The optimum dilutions of serum have been determined that will agglutinate all type strains of pneumococci and fail to cause any cross agglutination reactions when mixed with equal amounts of pneumococcus cultures and incubated for I hour at 37°C. They are a 1:20 dilution of Serum I, a 1:20 dilution of Seruifa II, and a 1:5 dilution of Serum III. For the diagnosis of Subgroup II pneumococci undiluted Type II serum is required. To obviate the other difficulties of the method commonly used a new method for the determination of types has been devised. It depends upon the fact that there is produced by the growth of the pneumococcus a soluble substance which is present in the peritoneal exudate of the mouse in sufficient quantity to give a specific precipitin reaction with the homologous immune serum. The precipitin method can be used in all instances in which the determination of types by the agglutination method is possible, and it possesses certain distinct advantages which make it available when the agglutination method is impracticable. It is of particular value as a time-saving device in those instances where the presence of other organisms together with the pneumococcus in the peritoneal exudate causes a delay of 18 hours or more before the type of pneumococcus can be definitely established. It is therefore recommended as the method of choice in all cases. If desired, both the agglutination and precipitin methods may be applied to the same specimen of peritoneal washings.     

Potential therapeutic uses for S-nitrosothiols.

The S-nitrosothiols (RSNOs) are thought to represent a circulating endogenous reservoir of nitric oxide (NO), and may have potential as donors of NO, distinct from currently used agents. They have the general formula RSNO, and naturally occurring examples include S-nitrosocysteine, S-nitrosoglutathione and S-nitrosoalbumin, in which R is an amino acid, polypeptide and protein respectively. RSNOs have anti-platelet properties, a theoretical role in the treatment of asthma and the potential to be used as agents to treat infectious diseases ranging from the common cold to AIDS. RSNOs are relatively unstable, being degraded to release NO and the corresponding disulphide. Their stability is influenced by the properties of the R group, heat, light, the presence of transition metal ions (in particular copper) and the presence of other thiols. RSNOs participate in transnitrosation reactions in which the -NO group is transferred to another thiol to form a more stable RSNO. Thus the stability of RSNOs in vivo is difficult to predict, and this has led to the development of more stable analogues, in which the properties of R are modified. Potential interactions of RSNOs include that with ascorbic acid (vitamin C), which enhances the ability of copper to catalyse their degradation. Transnitrosation reactions with thiol-containing enzymes can influence protein function, and the intracellular thiol glutathione, levels of which are influenced by many disease states, can also influence stability. Thus, although RSNOs have many theoretically useful properties, there are also many aspects of their biochemistry that need to be addressed before their therapeutic potential can be fully realized.     

PTH analogues and osteoporotic fractures

Importance of the field: At present there are two parathyroid hormone (PTH) analogues (PTH 1 – 34 and PTH 1 – 84) registered for the treatment of established osteoporosis in postmenopausal women (PTH 1 – 34 and PTH 1 – 84) and in men (PTH 1 – 34 only) who are at increased risk of having a fracture.

Areas covered in this review: The efficacy and safety of PTH 1 – 34 and PTH 1 – 84 in the management of osteoporosis is evaluated by reviewing published literature and presentations from scientific meetings through to 2010.

What the reader will gain: This review focuses on data on fracture risk reduction and safety endpoints of PTH analogues. The adverse reactions reported most are nausea, pain in the extremities, headache and dizziness.

Take home message: Exogenous PTH analogues, given as daily subcutaneous injections, stimulate bone formation, increase bone mass and bone strength, and improve calcium balance. In postmenopausal women with osteoporosis, PTH analogues reduced the risk of vertebral (PTH 1 – 34 and PTH 1 – 84) and non-vertebral fractures (only PTH 1 – 34). In men and women with glucocorticosteroid-induced osteoporosis, PTH 1 – 34 reduced the risk of vertebral fractures. In general, PTH analogues are well tolerated with an acceptable safety profile: they can be used for the prevention and treatment of fractures in postmenopausal women with severe, established osteoporosis.     

Allergic contact eczema caused by trifluorothymidine

Over the past years pyrimidine analogues have gained an important role within the concept of local therapy of viral infections, in particular in the field of ophthalmology. This report deals with a case of contact allergy to trifluorothymidine in a 15 year-old atopic patient. Since until now only a small number of contact allergic reactions have been reported, the halogenated pyrimidine analogues seem to be weak sensitizers. Cross reactivity within these substances may occur.     

Pharmacogenetic screening and therapeutic drugs.

BACKGROUND: Pharmacogenetics is the science of the influence of heredity on pharmacological response. ISSUES: The cost of severe adverse drug reactions in individuals has been estimated in the US alone to be in excess of US$4 billion. It has been argued that in a significant proportion of cases, the efficacy and toxicity profiles of drug therapy would be substantially improved in individuals if characteristics due to genetic variation were taken into account. Methods are now available, which make screening for susceptibility feasible. CONCLUSIONS: There are several therapeutic areas in which screening may give rise to significant improvements in outcome with cost-benefits to both the individual and the community. However, there is currently a lack of data on which cost-benefit analysis can be based. The challenge is to provide this information for new drugs, and for drugs with established therapeutic roles.     

Photooxidation of thiosaccharides mediated by sensitizers in aerobic and environmentally friendly conditions

A series of β-d-glucopyranosyl derivates have been synthesized and evaluated in photooxidation reactions promoted by visible light and mediated by organic dyes under aerobic conditions. Among the different photocatalysts employed, tetra-O-acetyl riboflavin afforded chemoselectively the respective sulfoxides, without over-oxidation to sulfones, in good to excellent yields and short reaction times. This new methodology for the preparation of synthetically useful glycosyl sulfoxides constitutes a catalytic, efficient, economical, and environmentally friendly oxidation process not reported so far for carbohydrates.

An environmentally friendly and simple sensitized photooxidation methodology to obtain glycosyl sulfoxides with outstanding chemoselectivity in aerobic conditions is described.