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Ornithine transcarbamylase (OTC) deficiency is an X-linked disorder that causes recurrent and life-threatening episodes of hyperammonemia. The clinical picture in heterozygous females is highly diverse and derives from the genotype and the degree of inactivation of the mutated X chromosome in hepatocytes. Here, we describe molecular genetic, biochemical, and histopathological findings in the livers explanted from two female patients with late-onset OTC deficiency. Analysis of X-inactivation ratios by DNA methylation-based assays showed remarkable intra-organ variation ranging from 46:54 to 82:18 (average 70:30, n = 37), in favor of the active X chromosome carrying the mutation c.583G>C (p.G195R), in the first patient and from 75:25 to 90:10 (average 82:18, n = 20) in favor of the active X chromosome carrying the splicing mutation c.663+1G>A in the second patient. The X-inactivation ratios in liver samples correlated highly with the proportions of OTC-positive hepatocytes calculated from high-resolution image analyses of the immunohistochemically detected OTC in frozen sections that was performed on total area > 5 cm 2 . X-inactivation ratios in blood in both female patients corresponded to the lower limit of the liver values. Our data indicate that the proportion of about 20-30% of hepatocytes expressing the functional OTC protein is not sufficient to maintain metabolic stability. X-inactivation ratios assessed in liver biopsies taken from heterozygous females with X-linked disorders should not be considered representative of the whole liver.

Renal hypouricemia is a rare heterogeneous inherited disorder characterized by impaired tubular uric acid transport with severe complications, such as acute kidney injury. Type 1 and 2 are caused by loss-of-function mutations in the SLC22A12 and SLC2A9 gene, respectively. A cohort of 881 randomly chosen ethnic Roma from two regions in Eastern Slovakia and two regions in the Czech Republic participated. Genomic DNA was isolated from buccal swabs and/or from blood samples. The c.1245_1253del and c.1400C>T genotypes were determined using polymerase chain reaction with allele-specific primers in a multiplex arrangement and/or direct sequencing of exon 7 and 9. Allele frequencies and genotypes were tested for Hardy-Weinberg equilibrium using the Chi-square test. 25 subjects were heterozygous and three were homozygous for the c.1245_1253del, while 92 subjects were heterozygous and two were homozygous for the c.1400C>T. Moreover, two participants were compound heterozygotes. Frequencies of the c.1245_1253del and c.1400C>T variants were 1.87 and 5.56 %, respectively. Our finding confirms an uneven geographical and ethnic distribution of SLC22A12 mutant variants. We found that the c.1245_1253del and c.1400C>T variants were present in the Czech and Slovak Roma population at unexpectedly high frequencies. Renal hypouricemia should be kept in mind during differential diagnostic on Roma patients with low serum uric acid concentrations.

Occurrence of carbapenemase-producing organisms, including New Delhi metallo-β-lactamase-1 (NDM-1) is increasingly reported worldwide. The aim of this study was to assess the distribution of carbapenemase producers among multidrug-resistant Gram-negative bacteria isolated from blood cultures. All carbapenem-resistant strains collected from December 2011 to December 2012 were analyzed. Presence of carbapenemases was assessed with combined disc test and Carba NP test followed by polymerase chain reaction for carbapenemase genes. Altogether, 30 strains were detected, of which 7 were positive for VIM (23.3%), 6 for NDM-1 (20%), 5 for IMP (16.7%), and KPC was present in one isolate (3.3%). Four Pseudomonas aeruginosa strains were found to produce more than one carbapenemase. We also present the case report of a patient with Acinetobacter baumannii ventilator-associated pneumonia, followed by sepsis due to Enterococcus faecalis and pan-resistant NDM-1-producing P. aeruginosa. Despite the inappropriate therapy, the patient was successfully treated. This is the first report of NDM-1-producing strains in Slovakia and it contributes to a number of studies mapping the distribution of carbapenemase producers in Europe.

Objective: To determine patterns of nasopharyngeal colonisation in HIV-positive children., Methods: Nasopharyngeal, nasal and ear swabs were prospectively taken from all children living in two paediatric nursing homes for HIV-positive orphans in Cambodia from 2004 to 2011., Results: A total of 882 swabs were taken, of which 586 tested positive for bacteria. Staphylococcus aureus was the most frequently isolated species (178 isolates; 30.4%) followed by Streptococcus pneumoniae (103 isolates; 17.6%) and Klebsiella pneumoniae (99 isolates; 16.9%). The rate of S. pneumoniae decreased in 2009 when a vaccination programme was introduced., Conclusions: The respiratory tract of HIV-positive children receiving highly active antiretroviral therapy is commonly colonised by S. aureus and S. pneumoniae, while other species normally found in the respiratory tract, such as Moraxella catarrhalis, are far less frequent., (© 2013 John Wiley & Sons Ltd.)

Objective: Infections involving the central nervous system have very serious consequences and affect thousands of people in Africa. Despite the availability of new antibiotics and vaccines, neuroinfections act as dangerous and life-threatening conditions. The most frequent neuroinfections which are of the greatest importance for public health systems are viral diseases (such as HIV, encephalitis, poliomyelitis, rabies), bacterial diseases (bacterial meningitis, neurological complications of leprosy and tuberculosis) and parasitic infections (cerebral malaria, sleeping sickness, trypanosomiasis, schistosomiasis, toxoplasmosis etc.)., Methods: A descriptive study to assess the occurrence of neuroinfections in two rural hospitals in Sudan (Mapuordit in Yirol and Gordim in Aweil) was performed in two periods of two years: (i) 2005-2006 and (ii) 2010-2011. We obtained data on patients from Mapuordit and from Gordim by studying their medical records., Results: Several cases of neuroinfections were observed during both periods; those were represented by tetanus, meningococcal meningitis, leprosy with neuropathy (altogether 442 patients) in Mapuordit. Also in Gordim, severe neuroinfections such as cerebral malaria were very rare (1 case), as well as tetanus (1 case), meningococcal meningitis (8 cases) and sleeping sickness (9 cases). However, the incidence of neuroinfections decreased from 44/1000 in 2005-2006 to 2/1000 in 2010-2011., Conclusions: Decreased incidence of serious neuroinfections (cerebral malaria, sleeping sickness, meningococcal meningitis) in Sudan may be related to improvement of effective therapeutic options, represented by (i) intermittent preventive therapy (IPT) for malaria, (ii) by suppression of sleeping sickness vectors and (iii) by better accessibility of antibiotics.

Objective: Many infections occurring in area of Sub-Saharan Africa are associated with more or less serious neurologic symptoms or complications. The aim of this study was to assess the incidence of selected infectious diseases in the equatorial part of Uganda and Kenya and to monitor potential neurological complications of these infections., Methods: The study was performed for May - August 2008. Patients suffering from cerebral malaria, AIDS, meningitis, typhoid, tuberculosis (TB), syphilis, leprosy, and trypanosomiasis patients were enrolled. Besides of standard examination, lumbar puncture (LP) and cerebrospinal fluid (CSF) examination was performed, and the occurrence of neurological disorders and sequellae was recorded and assessed., Results: Altogether 288 patients with neurological manifestation were enrolled. Malaria was the most prevalent disease in this study (102 cases, 35.42%), followed by typhoid (47 cases, 16.2%) and meningitis (38 cases, 13.2%). Leprosy and trypanosomiasis were only rarely detected (2.3% and 1.4%, respectively)., Conclusion: In malaria and HIV hyper-endemic area of rural Uganda, cerebral malaria is the leading tropical neuroinfection. Also, meningitis is still frequent probably due to insufficient access to vaccination.

Background: Parathyroid hormone-related protein (PTHrP) is an endothelial-derived vasoactive peptide. This study investigated whether bioactive PTHrP is locally released in a pressure-dependent way., Methods: A PTHrP antibody directed against the midregional part of PTHrP was used to analyze PTHrP in plasma samples. The biological activity of this PTHrP-like peptide was investigated in vitro. Plasma values were determined in samples from the left pulmonary artery and the arteria femoralis, taken under basal conditions and after the application of oxygen or iloprost to lower the pulmonary pressure. Twenty young patients (mean age 6.5 years), who were catheterized for an analysis of the reactivity of the pulmonary bed, were investigated. Endothelial function was investigated by acetylcholine responsiveness., Results: The antibody recognized a 30-kDa protein with in vitro PTHrP-like activity. In 11 patients (responders) with intact endothelial function, the PTHrP values determined in the left pulmonary artery were higher than those in the arteria femoralis. The local increase in the PTHrP concentration was reduced when either oxygen or iloprost lowered the pressure. Nine patients with endothelial dysfunction did not show any concentration gradients at any time (nonresponders)., Conclusions: The local concentration of bioactive PTHrP is increased in patients with pulmonary hypertension and normal endothelial function., (Copyright (c) 2007 S. Karger AG, Basel.)

In homocystinuria due to cystathionine beta-synthase (CBS) deficiency, vitamin B6 response has been linked to distinct mutations and ruled out for others. The splice site mutation c.1224-2A>C leading to the deletion of exon 12 is predominantly found in patients from Central Europe, where it has been found on in average 14% of mutant alleles. In this study we analyzed the clinical picture in 17 CBS deficient carriers of c.1224-2A>C. Homozygotes for c.1224-2A>C did not respond to vitamin B6, while in compound heterozygotes the response to vitamin B6 depended on the mutation on the second allele. Maximum likelihood analysis revealed one common haplotype of the c.1224-2A>C alleles. Additionally, we report the four novel CBS mutations c.451G>A (p.Gly151?), c.740_769del (p.Lys247_Gly256del), c.862G>C (p.Ala288Pro) and c.1135C>T (p.Arg379Trp). In summary, the data of this study suggest that the CBS c.1224-2A>C allele confers vitamin B6 nonresponsiveness and that this mutant allele came from a common ancestor., (Copyright 2004 Wiley-Liss, Inc.)

We screened adult and larval secretions and midden piles for the presence of Thelohania solenopsae spores to decipher potential sources for the horizontal transmission of the pathogen in fire ants. Hemolymph samples from both adult and larvae were also screened to rule out hemolymph contamination of samples. In adults, Thelohania spores were found in the crop and the fecal fluids, although only free spores were found in the fecal fluids of adults. In fourth instar larvae, both free and octospores were seen in midgut and the meconium samples. All of the midden pile samples had T. solenopsae spores of both types. Based on these results, we theorize that the pathogen may be horizontally transmitted within a colony by the removal and sharing of meconium of prepupating fourth instar larvae by adult workers and by the adult fecal droppings, and intercolonially by contamination of midden piles or brood raiding.

The in vitro generation of reactive oxygen species (ROS) in haemocytes of Galleria mellonella, Aporia crataegi, Dendrolimus sibiricus, Aglais urticae (Lepidoptera) and Gryllus bimaculatus (Orthoptera), was studied by the method of nitroblue tetrazolium (NBT) reduction. Formazan formation (product of NBT reduction) was observed in haemocytes of all the insects examined, except A. urticae. Lypopolysaccharide and zymosan reduced the number of NBT-positive cells after 1 h incubation and an increase was registered after 4 h incubation. The inhibitors of the respiratory chain enzyme (sodium azide) and melanogenesis (phenylthiourea) reduced formazan formation in nonactivated insect blood cells. No influence of sodium azide and phenylthiourea was found on the activated haemocytes. The results suggest that the generation of ROS in insect haemocytes occured as a result of processes such as respiration and melanization during phagocytosis and encapsulation.

Microsporidia (M), representatives of the phylum Microspora, make a world-wide distributed group of intracellular protists, parasitic in the vast number of hosts, from Protozoa to Primates. In their morpho-functional organization, both very primitive and extremely specialized features are seen definitely combined. Data available on RNA and DNA sequences suggest that M may be the most ancient eukaryotes. By the present, as many as 13 microsporidian species have been recognized as opportunistic pathogens in AIDS and transplant patients. Information about structural, transport and regulatory proteins of M, as well as on their enzymes is scarce, though it could serve as a basis for understanding pathogenicity of M and indicate some possible sites of relevant suppressive therapy. The present study persuaded two main goals: 1) to examine two ways of antigen preparation (from the infected organ and from the purified spores) and to evaluate their relation to the yield of the resulting antibodies; 2) to identify and localize new proteins with the help of the obtained antibodies by means of IFA, IEM and WB. Mice were immunized: 1) with dissolved proteins of heavily loaded with parasites fat bodies isolated from crickets Gryllus bimaculatus infected with Nosema grylli, and 2) with proteins of the purified spores of N. grylli. As a result two antisera were obtained. Antiserum 1 reacted predominantly with spore walls on IFA slides and ultrathin sections (IEM). It also reacted with a broad spectrum of parasite and host cell proteins on WB. Antiserum 2 recognized polar filaments and walls of discharged spores in IFA and IEM tests. It did not react with undischarged spores or fat bodies of uninfected crickets and gave a comparatively weak reaction with those of the infected hosts. Hybridization of spleen cells of immune mice with murine myeloma cells resulted in several hybridoma clones. They produced Mabs, 5 of which were tested by IFA, IEM and WB. Mab 1BF3 recognized 55 kDa protein connected with polar filaments as it was clearly suggested by IEM and IFA. Mab 1BD9 recognized 25, 34, 43 kDa proteins from the fraction of membrane bound proteins of spore walls, the sites of their interaction with antigens being marked with uneven fluorescence (IFA) and by gold precipitates on spore walls (IEM). Mab 1BB9 reacted with 36, 45, 65 and 75 kDa proteins, which belong mainly to the fraction of membrane-bound spore proteins, and gave a weak fluorescence associated with spores. Mab 2AB3 recognized 44 and 60 kDa proteins from the fraction of soluble spore proteins, and Mab 2AD4 acknowledged a single protein of 55 kDa from the same fraction. The obtained antibodies add to the existing microsporidian antibody bank and can be used for further work of isolation, description and sequencing the microsporidian proteins in order to understand eventually their functions.

The presence of 14 enzymes was investigated using purified spores of the microsporidian Nosema grylli from fat body of the crickets Gryllus bimaculatus. Glucose 6-phosphate dehydrogenase (EC 1.1.1.49), phosphoglucomutase (EC 5.4.2.2), phosphoglucose isomerase (EC 5.3.1.9), fructose 6-phosphate kinase (EC 2.7.1.11), aldolase (EC 4.1.2.13), 3-phosophoglycerate kinase (EC 2.7.2.3), pyruvate kinase (EC 2.7.1.40) and glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) were detected with activities of 15 +/- 1, 7 +/- 1, 1,549 +/- 255, 10 +/- 1, 5 +/- 1, 16 +/- 4, 6 +/- 1 and 16 +/- 2 nmol/min mg protein, respectively. Hexokinase (EC 2.7.1.1), NAD-dependent malate dehydrogenase (EC 1.1.1.37), malic enzyme (EC 1.1.1.40), lactate dehydrogenase (EC 1.1.1.27), alcohol dehydrogenase (EC 1.1.1.1) and succinate dehydrogenase (EC 1.3.99.1) were not detectable. These results suggest the catabolism of carbohydrates in microsporidia occurs via the Embden-Meyerhof pathway. Glycerol 3-phosphate dehydrogenase may reoxidize NADH which is produced by glyceraldehyde 3-phosphate dehydrogenase in glycolysis.