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BACKGROUND: Severe infections in the absence of secondary immunodeficiency can alert clinicians to single-gene inborn errors of immunity/primary immunodeficiency disorders (PIDDs). Mendelian susceptibility to mycobacterial disease (MSMD) is characterised by selective susceptibility to mycobacterial infections due to inborn errors in the interleukin 12-interferon gamma pathway. The South African (SA) burden of hyperendemic tuberculosis (TB) infection provides an interesting context for the study of MSMD. OBJECTIVES:  To evaluate whether severe, persistent, unusual or recurrent (SPUR) definitions of TB can be applied in the context of MSMD in SA. METHODS: This study is a retrospective review of an SA PIDD cohort. Patients aged 0 - 15 years with SPUR TB infections, assessed between 2013 and 2018, were identified using a proposed algorithm. HIV infection or other secondary causes for immunodeficiency were excluded. Basic investigations, then focused immunophenotyping and next-generation sequencing, were performed. RESULTS:  A total of 20 patients with a clinical diagnosis of MSMD were identified. A further two, forming part of a family cohort, had pathogenic variants but remain asymptomatic. Infection with Mycobacterium tuberculosis complex predominated (64%), while 27% had BCG infection or non-tuberculous mycobacteria (NTM) infection. Molecular analysis revealed pathogenic variants in 41% of patients with SPUR mycobacterial infection, mainly in those with BCG/NTM infection. CONCLUSIONS:  In the SA paediatric population, SPUR TB infections, particularly BCG/NTM, in the absence of secondary immunodeficiency, can alert to possible MSMD. The molecular diagnosis is pivotal, guiding disease classification and influencing clinical approach and management. The diagnosis is complex and requires a multidisciplinary approach with close collaboration between clinical immunologists, bioinformaticians, immunologists, clinical geneticists and genetic counsellors.

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A transfacultary seminar of students with a patient resulted in a theatre performance based on Goethe's "Faustus" demonstrating the patient's treatment marathon in our current health-care system. Core demands and results are (i) simple access to evidence-based patient information, (ii) communication skills in the selection and education of medical students. and (iii) active involvement of patients in the self-management of their illness.

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BACKGROUND: This clinical trial is focussed on the 12-month results of a new method for refractive correction, femtosecond lenticule extraction (FLEx). PATIENTS AND METHODS: In a prospective 6-month study 108 eyes were treated in Erfurt and Marburg. Both a flap and a lenticule of intrastromal corneal tissue were simultaneously cut utilising the Carl Zeiss Meditec AG (Jena, Germany) VisuMax femtosecond laser system. Thereafter, the lenticule was manually removed and the flap repositioned. 31 patients all treated in Erfurt volunteered for a 12-month follow-up. UCVA and BSCVA after 12 months, objective and manifest refraction as well as slit-lamp examination and late side effects were documented. RESULTS: 62 eyes of 31 patients were examined in this study. The spherical equivalent before surgery was -4.81 (± 1.16) D; after 12 months -0.15 (± 0.46) D were manifest. No significant change was observed after the 6 months control. Starting with UCVA of 0.12 (± 0.09) before surgery UCVA was 1.10 (± 0.26) after 12 months. In one patient corneal ectasia was diagnosed. All other patients did not have any late side effects. CONCLUSION: The stability of the correction of myopic astigmatism with the FLEx procedure is very good. The fact that one patient presented with a corneal ectasia demonstrates that one general problem of refractive corneal surgery persists with this new fs technology.

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On 25 September 2007, soybean leaves with lesions typical of soybean rust were found in two commercial fields located in Dallas County, Iowa. Growth stage of the infected soybean plants was near physiological maturity. Diagnosis of Phakopsora pachyrhizi on leaves was confirmed by morphological observation of uredinia and urediniospores and conventional PCR conducted by the Iowa State University Plant and Insect Diagnostic Clinic using P. pachyrhizi-specific primers Ppm1 and Ppa2 as described (1). Water blanks were used as negative controls in PCR testing. Leaves were collected from additional counties throughout Iowa and examined microscopically. Soybean rust was identified on leaves from 14 counties, almost all the counties sampled, ranging from far western to far eastern Iowa. The northernmost detection was at 42.9°N in Hancock County, which also is the northernmost detection of soybean rust in the continental United States so far. In a commercial field in Fremont County, in the southwestern corner of Iowa along the Missouri River, disease incidence was approximately 20% and disease severity was 5%. Observed disease incidence was 1 to 2% and severity was less than 1% from all other samples. Most uredinia were scattered on the leaves as single pustules or clustered in groups of three to five pustules. Pustules on some leaf samples were sporulating, depending on weather conditions at the time when samples were collected. Fresh urediniospores collected from the leaf samples were placed on glass slides with free water on the surface and incubated in a dew chamber under darkness for 8 h to test for germination. Germination rates ranged from 80 to 90%. Rust spores also were used to inoculate detached soybean trifoliate leaves, which were kept in a dew chamber under darkness for 12 h with water-soaked cotton wrapped around the petioles. Typical soybean rust pustules developed within 10 to 14 days after incubation. The detections of soybean rust in Iowa were consistent with the predictions of an aerobiological spore dispersal model, which predicted spore showers in central to western Iowa in August. Above normal wet weather in the Great Plains and Iowa may have favored the statewide disease occurrence. To our knowledge, this is the first report of soybean rust in Iowa. Reference: (1) R. D. Frederick et al. Phytopathology 92:217, 2002.

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Ceratocystis fimbriata is a widely distributed, plant pathogenic fungus that causes wilts and cankers on many woody hosts. Earlier phylogenetic analyses of DNA sequences revealed three geographic clades within the C. fimbriata complex that are centered respectively in North America, Latin America and Asia. This study looked for cryptic species within the North American clade. The internal transcribed spacer regions (ITS) of the rDNA were sequenced, and phylogenetic analysis indicated that most isolates from the North American clade group into four host-associated lineages, referred to as the aspen, hickory, oak and cherry lineages, which were isolated primarily from wounds or diseased trees of Populus, Carya, Quercus and Prunus, respectively. A single isolate collected from P. serotina in Wisconsin had a unique ITS sequence. Allozyme electromorphs also were highly polymorphic within the North American clade, and the inferred phylogenies from these data were congruent with the ITS-rDNA analyses. In pairing experiments isolates from the aspen, hickory, oak and cherry lineages were interfertile only with other isolates from their respective lineages. Inoculation experiments with isolates of the four host-associated groupings showed strong host specialization by isolates from the aspen and hickory lineages on Populus tremuloides and Carya illinoensis, respectively, but isolates from the oak and cherry lineages did not consistently reveal host specialization. Morphological features distinguish isolates in the North American clade from those of the Latin American clade (including C. fimbriata sensu stricto). Based on the phylogenetic evidence, interfertility, host specialization and morphology, the oak and cherry lineages are recognized as the earlier described C. variospora, the poplar lineage as C. populicola sp. nov., and the hickory lineage as C. caryae sp. nov. A new species associated with the bark beetle Scolytus quadrispinosus on Carya is closely related to C. caryae and is described as C. smalleyi.

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The plant pathogenic fungus Ceratocystis fimbriata f. platani attacks Platanus species (London plane, oriental plane and American sycamore) and has killed tens of thousands of plantation trees and street trees in the eastern United States, southern Europe and Modesto, California. Nuclear and mitochondrial DNA fingerprints and alleles of eight polymorphic microsatellite markers of isolates of C. fimbriata from these regions delineated major differences in gene diversities. The 33 isolates from the eastern United States had a moderate degree of gene diversity, and unique genotypes were found at each of seven collection sites. Fingerprints of 27 isolates from 21 collection sites in southern Europe were identical with each other; microsatellite markers were monomorphic within the European population, except that three isolates differed at one locus each, due perhaps to recent mutations. The genetic variability of C. fimbriata f. platani in the eastern United States suggests that the fungus is indigenous to this region. The genetic homogeneity of the fungus in Europe suggests that this population has gone through a recent genetic bottleneck, perhaps from the introduction of a single genotype. This supports the hypothesis that the pathogen was introduced to Europe through Naples, Italy during World War II on infected crating material from the eastern United States. The Californian population may also have resulted from introduction of one or a few related genotypes because it, too, had a single nuclear and mitochondrial genotype and limited variation in microsatellite alleles.

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Recently it was shown that extracellular ATP, acting through purinergic receptors, has many physiological functions, including opening of Ca(2+)-ion channels, activation and mediation of signal transduction mechanisms as well as activation of the pain sensation. Since electrical stimulation is also known to affect many signal transduction processes as well as the alleviation of pain, we hypothesized that electric stimulation may affect the extracellular release of ATP. We investigated the effects of a small DC electric field (10(1)--10(2) V m(-1) range and with frequencies below 150 Hz) on the release of ATP in vitro (HeLa cells), and on the levels of ATP in vivo (the plasma of healthy volunteers). In HeLa cells ATP release was increased 50 fold, while the total amount of ATP in the cells was increased by 163%. In the plasma a significant decrease (P<0.05) in ATP concentration was seen after electrical stimulation, in all the volunteers. The small DC electric field also affected the cAMP signal transduction system in vitro (HeLa cells and human lymphocytes) and in vivo (human plasma). Decreased levels of cAMP (P<0.05) were seen in HeLa cells and increased levels of cAMP (P<0.05) in isolated human lymphocytes. The cAMP levels in the plasma of the electrically treated volunteers were lower than control values. These results show that the frequency, waveform and signal strength of the applied electric field are suitable for effecting measurable changes on signal transduction in vitro and in vivo.

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Successful treatment of various medical complaints with an applied electric field has been reported over the years. The identities of the cellular mechanisms that are influenced by this type of treatment and facilitate the positive effects, remain elusive. A study of many in vitro and in vivo reports revealed that the beneficial effects can be attributed to the activation of membrane proteins, and specifically proteins involved in signal-transduction mechanisms. Not only may the proteins be affected but it is now well established that enhanced Ca(2+)influx, observed to follow electric stimulation of cells, also contributes to many calcium-dependent cellular processes which can be linked to the therapeutic effects discussed in this paper. An hypothesis of the physical changes caused by an applied, relatively small (10(3)to 10(4)V m(-1)range), electric field with low to moderate frequency (below 150 Hz), is postulated.

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[3H]uridine and [3H]orotic acid were equally utilized for labelling of RNA in mouse liver. Incorporation of [3H]cytidine was 2-3 times as high as that of [3H]-labelled uridine or orotic acid. These results differ from findings in rat liver, where both cytidine and orotic acid are better utilized for RNA labelling than is uridine. The ratio between liver RNA [3H]-activity and volatile [3H]-activity was 2, 3 and 13, respectively, at 300 min after injection of labelled uridine, orotic acid and cytidine, indicating an efficient chanelling of cytidine into liver anabolic pathways.

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The metabolism of [5-3H]uridine and the incorporation of the precursor into liver RNA was studied in developing (13-day-old) and adult (45-day-old) mice. Different time-courses of labelling and increased amounts of labelled catabolic products of uridine were found in liver and blood of developing mice compared with adult animals. This is suggested to be a consequence of enlarged metabolite pools resulting from a lower total amount of uracil-degrading enzymes in the developing mice. The labelling of the uracil nucleotides was decreased in the developing liver. However, in spite of a lower specific radioactivity of UTP, the RNA-specific radioactivity of developing liver was increased compared with adult liver. Also the labelling of liver RNA with [6-14C]orotic acid was found to be increased in developing mice, thus indicating a higher rate of RNA synthesis in these animals. A more pronounced difference in liver RNA labelling between the developing and the adult mice obtained with the use of [14C]orotic acid than with [3H]uridine may suggest that the de novo pathway, relative to the salvage pathways, is more important in developing than in adult liver.

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The synthesis of RNA during mouse liver regeneration was studied by two different methods at 24 and 48 h after partial hepatectomy. Total chromatin-bound RNA polymerase activity showed an increase of 32% at 24 h after partial hepatectomy. At 48 h a slight increase in total activity was also observed in regenerating liver, but the difference was not significant. The increase in total RNA polymerase activity was due to a rise in RNA polymerase I plus III activity. This enzyme activity was increased at both 24 and 48 h. The increase was 57% at 24 h and 51% at 48 h. When [methyl-14C]methionine was used for labelling of methyl groups in rRNA, there was an increased specific radioactivity of this class of RNA at both 24 h and 48 h. The increases were 263 and 103% at 24 and 48 h respectively. Thus both methods revealed an increased synthesis of rRNA during mouse liver regeneration. The results are discussed in relation to previous results from this laboratory [Yngner, Carlberg, Lewan & Engelbrecht (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1069-1074; Yngner, Engelbrecht, Lewan & Annerfeldt (1979) Biochem. J. 178, 1-8; Yngner, Bengtsson, Carlberg, Engelbrecht & Wieslander (1980) Exp. Cell. Biol. 48, 393-403], which have shown that the incorporation of orotic acid or uridine into RNA is not increased in mouse liver regenerating after partial hepatectomy.

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The effects of thymidine (TdR) co-administration on the cytotoxicity and incorporation of 5-fluorouracil (5-FU) into RNA of various tissues was studied in rats bearing an ascites hepatoma (AH 130). The role of pyrimidine degradation in determining the modulating effects of TdR on the formation of FU-RNA was studied in hepatocytes and AH 130 cells in vitro. TdR (500 mg/kg) potentiated the antitumour effect of 5-FU (150 mg/kg) and also increased host toxicity as judged by changes in body weight. TdR given alone did not significantly affect tumour growth and body weight gain. Examination of the effect of TdR on the incorporation of 5-FU into RNA revealed a differential modulation of RNA-directed toxicity in different tissues. Incorporation of 5-FU into RNA in tumour and bone marrow was increased 2- and 4-fold, respectively. In spleen and kidney the incorporation increased by approximately 50%, but the values did not reach statistical significance. In contrast, the incorporation into RNA of liver and intestinal mucosa was decreased to ca 35% of the control. TdR at concentrations of 40 microM-40 mM progressively inhibited the degradation of 5-FU and decreased the incorporation of 5-FU into RNA of hepatocytes in vitro. In AH 130 cells in vitro TdR did not significantly influence the metabolism of 5-FU and the incorporation into RNA. These results demonstrate that the enhanced incorporation of 5-FU into tumour RNA in vivo after pretreatment with TdR is related not to local effects on the tumour cells but rather to an increased bioavailability of the drug. Although co-administration of TdR did not selectively enhance the antitumour effect of 5-FU, a differential toxicity in host tissues was indicated by the modulated incorporation of 5-FU into RNA.

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This study addresses the question whether urinary polyamine excretion is related to cell death or cell proliferation. CCl4 intoxication of the rat was used as the experimental model. Treatment with CCl4, a hepatotoxic haloalkane, produces an initial phase of liver cell death succeeded by a regenerative phase of growth, during which the liver is restored. The highest rate of putrescine (and spermidine) excretion occurred during the first 24 hr of CCl4 intoxication and coincided with the period of maximum liver damage. During subsequent liver regeneration the rate of excretion of both polyamines decreased.

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The mouse liver revealed no increased incorporation of [14C]-orotic acid into either the total acid-soluble fraction, the uridine triphosphate or the RNA at 6 and 24 h after partial hepatectomy. In regenerating mouse and rat liver, the concentration of adenosine triphosphate was decreased 15-20% at 6 h, but was in the same range as that of the controls at 24 h. The adenosine monophosphate concentration of mouse liver increased 4-fold and 2-fold at these times after partial hepatectomy, respectively. The results indicate no direct relationship between the energy metabolism and the uptake and incorporation of orotic acid into RNA of regenerating liver. The activity of mouse plasma lactate dehydrogenase 5 (LDH 5) was increased 12-fold at 6 h and 5-fold at 24 h after partial hepatectomy. In rat, the LDH 5 activity was increased 2-fold at 6 h but was not different from that of the controls at 24 h. An increased leakage of LDH 5, possibly related to the decreased energy content of the liver, was thus revealed by the partially hepatectomized mice.

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The uptake and utilization of [6-14C]orotic acid for UTP and RNA synthesis were studied in rat and mouse liver at 24 h after partial hepatectomy. Rat liver concentrated radioactivity relative to blood several-fold better than did mouse liver after both sham-operation and partial hepatectomy. The results showed that in mouse liver, contrary to rat liver, the orotic acid uptake was not increased after the partial hepatectomy. In rat liver, the precursor uptake and the labeling of UTP increased by about 75% whereas the specific radioactivity of RNA increased 2 to 3-fold after the operation, thus indicating an increased RNA synthesis. Mouse liver showed no increased [14C]orotic acid uptake or labeling of UTP or RNA at 24 h after partial hepatectomy.

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The balance between anabolism and catabolism of [5-(3)H]uridine was studied in the mouse after partial hepatectomy. Labelling of RNA and UDP-glucose was determined and evaluated in relation to changes in the specific radioactivity of UTP. The amounts of labelled catabolic products of uridine were increased several-fold in liver and blood after partial hepatectomy. The specific radioactivity of RNA decreased to about 60% of the control value at 6h and was in the same range as that of control liver at 24h after operation. Decreased labelling of RNA and UDP-glucose was attributable to decreased specific radioactivity of UTP. No changes in the size of the UTP pool or in the balance between uridine anabolism and catabolism were found that could explain the decreased specific radioactivity of UTP. Rather, the alterations in the labelling of this metabolite induced by the partial hepatectomy may be related to decreased phosphorylating capacity in the liver cells and/or dilution of the labelled precursor in an expanded uridine pool. The enhanced amounts of uridine catabolic products in liver and blood were probably a consequence of accumulation and altered incorporation of the metabolites from the blood into the liver cells. Despite the increased amounts of labelled catabolic products and the decreased labelling of RNA, the results reported here actually suggest decreased uridine catabolism and slightly increased RNA synthesis in mouse liver after partial hepatectomy. The results stress the importance of proper controls in determination of nucleic acid synthesis and in metabolic studies by use of labelled precursors.

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[14C]Orotic acid was rapidly distributed in blood after both i.p. and s.c. injection but was not completely absorbed from the peritoneal cavity until 20 min after injection. S.c. injection should be an acceptable alternative to i.p. injection although the incorporation into the liver acid soluble- and RNA-fractions was somewhat delayed after the s.c. injection.

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