Tumor vascular signals in renal masses: detection with Doppler US

The vascularity of 49 renal masses (26 malignant and 23 benign lesions) was investigated with duplex Doppler ultrasound. Doppler signals obtained at the margins of renal masses were defined as "tumor signals" when the Doppler-shifted frequency of the lesion exceeded the frequency shift in the ipsilateral main renal artery. These exceeded 2.5 kHz with a 3-MHz insonating frequency. Among the 26 renal masses that subsequently proved to be malignant, tumor signals were obtained in 15 of 18 (83%) untreated renal cell carcinomas, in three of four Wilms tumors, and in two patients with metastases to the kidney, but not in the one patient with lymphoma. None of the 23 benign renal masses demonstrated tumor signals. Tumor vascularity in malignant lesions gives rise to abnormal, high-velocity, Doppler-shifted signals that can help in the differential diagnosis of renal masses.     

Viral dynamics and their relations to baseline factors and longer term virologic responses in treatment-naive HIV-1-infected patients receiving abacavir in combination with HIV-1 protease inhibitors

From a study of 71 HIV-1-infected patients receiving abacavir in combination with 1 of 5 different HIV-1 protease inhibitors (indinavir, ritonavir, saquinavir, nelfinavir, or amprenavir), we found that the baseline HIV-1 RNA levels were highly predictive of the viral decay rates. The baseline HIV-1 RNA levels were negatively correlated with the first phase viral decay rates (r = -0.77, P < 0.001) and positively correlated with the second phase viral decay rates (r = 0.68, P < 0.001). In addition, the first phase viral decay rate was positively correlated with CD4+ cell increases. No significant correlation was found between viral decay rates and longer term (24 weeks) virologic responses, and no difference in viral decay rates was found among the 5 study regimens. These data suggest that the potency of the 5 treatment regimens was similar and was not predictive of long-term virologic failure.     

Protein kinase C isotypes theta, delta and eta in human lymphocytes: differential responses to signalling through the T-cell receptor and phorbol esters.

The repertoire of novel and atypical protein kinase C (PKC) isotypes present in human T cells and their subcellular localization have not been fully characterized. We detected calcium-independent PKC activity in whole cell fractions from unstimulated peripheral blood lymphocytes (PBL). Towards an understanding of the role of PKC isoforms in lymphocyte activation we have studied the expression of calcium-independent PKC isoforms theta, delta and eta in PBL. With isoformspecific antibodies we detected the presence of PKC theta and delta in whole cell fractions from unstimulated human PBL by Western blot analysis. In addition, immunocytochemical analysis confirmed the presence of the novel PKC isoform PKC eta in PBL. Using immunocytochemistry, PKC theta, delta and eta had distinct patterns of redistribution following activation by phorbol myristate acetate (PMA). However, signalling through the T-cell receptor (TCR) did not appear to induce such changes in isoenzyme redistribution. These findings indicate that activation of lymphocytes either through the TCR-CD3 complex or with PMA induces different signalling pathways with respect to calcium-independent isoenzymes. Signalling through receptors other than the CD3 complex may be involved in activation of these isotypes.     

Detection of reactive oxygen species (ROS) and apoptosis in human fragmented embryos

In human in-vitro fertilization (IVF)-embryo transfer, the in-vitro culture environment differs from in-vivo conditions in that the oxygen concentration is higher, and in such conditions the mouse embryos show a higher concentration of reactive oxygen species (ROS) in simple culture media. ROS are believed to cause damage to cell membranes and DNA fragmentation in somatic cells. This study was conducted to ascertain the level of H2O2 concentration within embryos and the morphological features of cell damage induced by H2O2. A total of 62 human oocytes and embryos (31 fragmented, 15 non-fragmented embryos, 16 unfertilized oocytes) was obtained from the IVF-embryo transfer programme. The relative intensity of H2O2 concentrations within embryos was measured using 2',7'-dichlorodihydrofluorescein diacetate by Quanti cell 500 fluorescence imaging and DNA fragmentation was observed with transmission electron microscopy and an in-situ apoptosis detection kit. The H2O2 concentrations were significantly higher in fragmented embryos (72.21 +/- 9.62, mean +/- SEM) compared to non-fragmented embryos (31.30 +/- 3.50, P < 0.05) and unfertilized oocytes (30.75 +/- 2.67, P < 0.05). Apoptosis was observed only in fragmented embryos, and was absent in non-fragmented embryos. Electron microscopic findings confirmed apoptotic bodies and cytoplasmic condensation in the fragmented blastomeres. We conclude that there is a direct relationship between increased H2O2 concentration and apoptosis, and that further studies should be undertaken to confirm these findings.      

Lipophosphoglycan expression and virulence in Ricin-resistant variants of Leishmania major

Lipophosphoglycan (LPG) of Leishmania is a polymorphic molecule comprising an alkylglycerol anchor, a conserved oligosaccharide core and a species-specific polymer of oligosaccharide repeats joined by phosphodiester bonds. This molecule, together with the membrane polypeptide gp63, has been implicated as a parasite receptor for host macrophages. To examine the role of LPG in parasite infectivity glycosylation variants of Leishmania major were generated by chemical mutagenesis of a virulent cloned line V121 and variants with modified LPG selected using the galactose-specific lectin Ricinus communis II (RCA II). Twenty RCA II-resistant primary clones were generated. Analysis of LPG profile by immunoblotting using LPG-specific monoclonal and polyclonal antibodies revealed that some of the clones were LPG-deficient. Three clones that did not bind any LPG-specific antibodies but expressed normal levels of the M_r 63 000 glycoprotein (gp63), a second parasite receptor for host, were chosen for detailed studies.All three clones expressed, at least to some extent, a surface molecule which could be labeled by mild periodate oxidation and sodium borotritide and behaved like LPG by hydrophobic interaction chromatography. All clones also bound a well-characterized monoclonal antibody L157 directed to the core oligosaccharide of LPG, but did not bind another monoclonal antibody, CA7AE, to an epitope on a repeating unit shared by Leishmania donovani and L. major LPG. A third monoclonal antibody, 5E6, recognizing LPG on the surface of wild-type V121 promastigotes bound only to RCA II-resistant clone 3A2-C3 and was restricted to an internal structure. The LPG molecule that this clone expressed was a form of LPG by its chromatographic behavior and by its monosaccharide and alkylglycerol composition. Clone 3A2-C3 was the only one to infect mice in vivo and survive in macrophages in vitro, albeit at a much reduced rate compared to wild-type V121 promastigotes. The data suggest that some form of LPG may be necessary to ensure parasite infectivity.     

Human brucellosis in the Mid-West 2002-3

Human brucellosis remains a serious public health issue in Ireland. Clinical notifications in the Mid-Western Area (HSE-MWA) underestimate the burden of illness and attendant morbidity in the region. The diagnosis of acute and chronic human brucellosis depends on the clinical evidence and the results from laboratory serological testing or culture on rare occasion. This study examined the clinical evidence behind locally defined serological "positives" in the HSE-MWA from 2002 to 2003. Ninety cases were detected in 2002 and 31 in 2003. While sampling bias is likely to be present, aspects of brucellosis in Ireland were confirmed. Middle-aged males were most commonly affected. The majority of cases were linked to farming or veterinary practice. Symptoms such as sweats, fever and weight loss were commonly associated with acute brucellosis infection while malaise was common in acute and chronic brucellosis. A clear definition of what is notifiable is needed. Surveillance systems must appreciate the importance of both clinical and laboratory evidence to classify confirmed or probable brucellosis as paired sera were not common. Public health authorities must follow-up the clinical aspects for accurate national statistics. General practitioners in the Mid-West appear to be vigilant regarding brucellosis in their patients. Regional zoonoses committees are useful in monitoring disease prevalence in human and animal populations without compromising confidentiality.     

Optimization of peptide linker length in production of MHC class II/peptide tetrameric complexes increases yield and stability,and allows identification of antigen-specific CD4+T cells in peripheral blood mononuclear cells

Reliable, efficient systems for producing soluble HLA-DR molecules, suitable for multimerization and use as staining reagents, have proved elusive. We found that the addition of a flexible linker between peptide and N terminus of the DRB1*0101-chain (Crawford, F., Kozono, H., White, J., Marrack, P. and Kappler, J., Immunity 1998. 8: 675-682.), results in greater in vitro folding efficiency of Escherichia coli-expressed alpha- and beta-chains, and increases both the yield and stability of the DRA1*0101/DRB1*0101/peptide complexes. Although a 10-amino acid linker functioned efficiently for a 20mer epitope from HIV p24, a longer linker was required to produce a DR1 MHC class II tetramer with the influenza hemagglutinin epitope (HA(306-318)). The DR1-HA tetramer was able to stain positively over 98% of a specific clone (HA 1.7) with only a brief 30-min incubation. The tetrameric complexes detected clone cells diluted into PBMC, with high sensitivity, coupled with low background staining in CD4(+) cells. It was possible to detect antigen-specific CD4(+) T cells within a population of PBMC stimulated with the HA peptide. This demonstrates the potential to monitor CD4(+) T cell responses in peripheral blood in a number of clinical scenarios.     

Adult congenital heart disease

Continued advances in the understanding and management of congenital heart disease (CHD) mean that over 90% of children born with CHD now survive to adulthood. This in turn results in greater numbers of adult patients presenting for medical and surgical care at non-specialist centres. A simple classification of adult congenital heart disease (ACHD) according to complexity can help clinicians to understand the implications of the specific cardiac anomaly encountered. Issues relating to the conduct of anaesthesia in ACHD patient include careful attention to euvolaemia, the preservation of sinus rhythm and cardiac output, and in complex patients, manipulating the balance between systemic and pulmonary blood flows. Additionally, effective antibiotic prophylaxis and the prevention of either excessive bleeding or thromboembolism are vitally important. It should not be forgotten that although many patients with simple or repaired cardiac lesions may be very well managed in a non-specialist unit, those with Eisenmenger’s syndrome or severe pulmonary hypertension have an extremely high risk of death in the perioperative period, and in all but life-threatening situations should always be managed within specialist centres.     

Subtype‐specific differences in transmission cluster dynamics of HIV‐1 B and CRF01_AE in New South Wales,Australia

IntroductionThe human immunodeficiency virus 1 (HIV‐1) pandemic is characterized by numerous distinct sub‐epidemics (clusters) that continually fuel local transmission. The aims of this study were to identify active growing clusters, to understand which factors most influence the transmission dynamics, how these vary between different subtypes and how this information might contribute to effective public health responses.MethodsWe used HIV‐1 genomic sequence data linked to demographic factors that accounted for approximately 70% of all new HIV‐1 notifications in New South Wales (NSW). We assessed differences in transmission cluster dynamics between subtype B and circulating recombinant form 01_AE (CRF01_AE). Separate phylogenetic trees were estimated using 2919 subtype B and 473 CRF01_AE sequences sampled between 2004 and 2018 in combination with global sequence data and NSW‐specific clades were classified as clusters, pairs or singletons. Significant differences in demographics between subtypes were assessed with Chi‐Square statistics.ResultsWe identified 104 subtype B and 11 CRF01_AE growing clusters containing a maximum of 29 and 11 sequences for subtype B and CRF01_AE respectively. We observed a > 2‐fold increase in the number of NSW‐specific CRF01_AE clades over time. Subtype B clusters were associated with individuals reporting men who have sex with men (MSM) as their transmission risk factor, being born in Australia, and being diagnosed during the early stage of infection (p < 0.01). CRF01_AE infections clusters were associated with infections among individuals diagnosed during the early stage of infection (p < 0.05) and CRF01_AE singletons were more likely to be from infections among individuals reporting heterosexual transmission (p < 0.05). We found six subtype B clusters with an above‐average growth rate (>1.5 sequences / 6‐months) and which consisted of a majority of infections among MSM. We also found four active growing CRF01_AE clusters containing only infections among MSM. Finally, we found 47 subtype B and seven CRF01_AE clusters that contained a large gap in time (>1 year) between infections and may be indicative of intermediate transmissions via undiagnosed individuals.ConclusionsThe large number of active and growing clusters among MSM are the driving force of the ongoing epidemic in NSW for subtype B and CRF01_AE.     

The effect of zinc and vitamin C supplementation on the immune status of patients with Crohn's disease

The immune status of 29 patients with Crohn's disease given oral supplements of Vitamin C, zinc or placebo for three-week periods was studied. Collectively, the patients showed T-cell hyporesponsiveness, as assessed by phytohaemagglutinin stimulation, which was significantly improved by Vitamin C. Both monocyte function, as assessed by latex phagocytosis, and pan T-Cell number were significantly reduced and were not influenced by supplementation. Humoral immunity, assessed by pokeweed mitogen-induced immunoglobulin synthesis, was normal and remained unchanged. Vitamin C supplements improved T-cell function in Crohn's disease, whereas neither Vitamin C nor zinc had a measurable effect on humoral immunity.