Activation of immunoglobulin control elements in trasgenic mice

To assess the role interleukins and mitogens play in regulating immunoglobulin (Ig) gene expression via the Ig enhancer and promoter, transgenic mice carrying two different Ig gene regulatory regions were generated. One, EkCAT, contains the Ig heavy chain enhancer (E) and the light chain promoter driving the chloramphenicol acetyltransferase (CAT) gene. In the other, EkCAT, CAT is under the control of the promoter alone. E and relative activity were assessed by CAT assay. In EkCAT mice, low CAT expression was consistently found in spleen, bone marrow, mesenteric lymph node, and thymus but not in brain, lung, or kidney. In EkCAT mice, CAT expression was detectable just above background in lymphoid tissues, suggesting a basic level of tissue specificity in the absence of the enhancer. Whole spleen cell cultures prepared from the mice were treated with lymphokines and mitogens. Lipopolysaccharide (LPS), concanavilin A (Con A), interleukin 6 (IL-6), and interferon- (IFN-) increased CAT expression to varying extents in cells derived from EkCAT mice but not in spleen cells prepared from EkCAT mice. Thus, the presence of E, in addition to the promoter, is essential for the stimulation of CAT expression mediated by these factors. B cells from EkCAT mice were separated by density into populations of small and large cells. In untreated small B cells, no CAT expression was detected and only addition of LPS resulted in an increase in CAT expression. In large B cells, CAT was expressed at a low level without addition of exogenous factors. Incubation with LPS, IL-6, Con A and IFN- caused CAT expression to increase several-fold. This transgenic system provides a means to identify exogenous factors that activate Ig enhancers and promoters.This work has been submitted in partial fulfillment of the requirements for the doctoral degree from the George Washington University.     

Electron microscopical-immunocytochemical evidence of ecdysteroids in the prothoracic gland of Galleria mellonella

Summary Fixation of prothoracic glands of Galleria mellonella with a solution containing saponin permits immunocytochemical staining of the entire gland. By this means ecdysteroids were demonstrated electron microscopically to be present in the hyaloplasm and microtubules.Supported by Sächsische Akademie der Wissenschaften zu Leipzig     

Ultrastructure of the prothoracic gland cells of the last instar of Galleria mellonella in relation to the state of development

The prothoracic glands of the last instar of Galleria mellonella undergo characteristic alterations of their cellular fine structure closely related to cellular activity. During progressive secretory activity of the gland cells there are extensive plasmalemmal infoldings and formation of a pronounced lacunar system. Mitochondria of the active cell phase are characterized by a specific increase in size and paler colour of the matrix. In contrast to the alterations, nuclei, ER and Golgi cisterns do not undergo any submicroscopic changes during the different phases of cellular activity. The relationship between the substructural phenomena and the specific phases of cellular activity are discussed.     

Increased HEV Seroprevalence in Patients with Autoimmune Hepatitis

Background

Hepatitis E virus (HEV) infection takes a clinically silent, self-limited course in the far majority of cases. Chronic hepatitis E has been reported in some cohorts of immunocompromised individuals. The role of HEV infections in patients with autoimmune hepatitis (AIH) is unknown.

Methods

969 individuals were tested for anti-HEV antibodies (MP-diagnostics) including 208 patients with AIH, 537 healthy controls, 114 patients with another autoimmune disease, rheumatoid arthritis (RA), and 109 patients with chronic HCV- or HBV-infection (HBV/HCV). Patients with AIH, RA and HBV/HCV were tested for HEV RNA. HEV-specific proliferative T cell responses were investigated using CFSE staining and in vitro stimulation of PBMC with overlapping HEV peptides.

Results

HEV-antibodies tested more frequently positive in patients with AIH (n = 16; 7.7%) than in healthy controls (n = 11; 2.0%; p = 0.0002), patients with RA (n = 4; 3.5%; p = 0.13) or patients with HBV/HCV infection (n = 2; 2.8%; p = 0.03). HEV-specific T cell responses could be detected in all anti-HEV-positive AIH patients. One AIH patient receiving immunosuppression with cyclosporin and prednisolone and elevated ALT levels had acute hepatitis E but HEV viremia resolved after reducing immunosuppressive medication. None of the RA or HBV/HCV patients tested HEV RNA positive.

Conclusions

Patients with autoimmune hepatitis but not RA or HBV/HCV patients are more likely to test anti-HEV positive. HEV infection should been ruled out before the diagnosis of AIH is made. Testing for HEV RNA is also recommended in AIH patients not responding to immunosuppressive therapy.     

Kinetik des Aufbaus einer Proteinmembran. Untersuchungen an Gasvakuolen der blaugrünen Alge Microcystis aeruginosa

In wachsenden Kulturen der blaugrünen Alge Microcystis aeruginosa nimmt die Anzahl der Gasvakuolen pro Zelle ab und erreicht bei einer Zellzahl von 1,2 × 10⁷ pro ml eine Zahl von 4,5 × 10³ Vakuolen pro Zelle. Die Gasvakuolen können in den Zellen durch Ultraschall zerstört werden, danach regenerieren sie. In beschallten Kulturen wurde die Zeitabhängigkeit der Gasvakuolenbildung untersucht, indem die Anzahl und die Größe der neugebildeten Vakuolen bestimmt wurde. Innerhalb der ersten halben Stunde nach Ultraschallbehandlung werden 120 Gasvakuolen pro Zelle gebildet, nach 24 Stunden enthält jede Zelle durchschnittlich 2300 Vakuolen. Die Größe der Gasvakuolen nimmt ebenfalls mit der Zeit zu. In der ersten halben Stunde erreichen die bis dahin isodiametrischen Vakuolen eine Größe von 70 nm, dann wachsen sie nur noch in die Länge und haben 24 Stunden nach Ultraschallbehandlung eine durchschnittliche Länge von 286 nm. Rifampin und Chloramphenicol bewirken eine teilweise Hemmung der Gasvakuolenbildung. DCMU und Hydroxylamin blockieren die Vakuolenbildung völlig. Auch im Dunkeln werden keine Gasvakuolen gebildet     

Determinants of sustained viral suppression in HIV-infected patients with self-reported poor adherence to antiretroviral therapy

Background

Good adherence to antiretroviral therapy (ART) is critical for successful HIV treatment. However, some patients remain virologically suppressed despite suboptimal adherence. We hypothesized that this could result from host genetic factors influencing drug levels.

Methods

Eligible individuals were Caucasians treated with efavirenz (EFV) and/or boosted lopinavir (LPV/r) with self-reported poor adherence, defined as missing doses of ART at least weekly for more than 6 months. Participants were genotyped for single nucleotide polymorphisms (SNPs) in candidate genes previously reported to decrease EFV (rs3745274, rs35303484, rs35979566 in CYP2B6) and LPV/r clearance (rs4149056 in SLCO1B1, rs6945984 in CYP3A, rs717620 in ABCC2). Viral suppression was defined as having HIV-1 RNA <400 copies/ml throughout the study period.

Results

From January 2003 until May 2009, 37 individuals on EFV (28 suppressed and 9 not suppressed) and 69 on LPV/r (38 suppressed and 31 not suppressed) were eligible. The poor adherence period was a median of 32 weeks with 18.9% of EFV and 20.3% of LPV/r patients reporting missed doses on a daily basis. The tested SNPs were not determinant for viral suppression. Reporting missing >1 dose/week was associated with a lower probability of viral suppression compared to missing 1 dose/week (EFV: odds ratio (OR) 0.11, 95% confidence interval (CI): 0.01–0.99; LPV/r: OR 0.29, 95% CI: 0.09–0.94). In both groups, the probability of remaining suppressed increased with the duration of continuous suppression prior to the poor adherence period (EFV: OR 3.40, 95% CI: 0.62–18.75; LPV/r: OR 5.65, 95% CI: 1.82–17.56).

Conclusions

The investigated genetic variants did not play a significant role in the sustained viral suppression of individuals with suboptimal adherence. Risk of failure decreased with longer duration of viral suppression in this population.     

In Vitro Synthesis of Adenovirus Core Proteins

mRNA extracted from polysomes of KB cells at late stages of productive infection with adenovirus type 2 was translated in a cell-free system derived from Krebs II ascites cells. Two of the polypeptides obtained in this reaction corresponded to the adenovirus core protein V and the precursor to core protein VII. The following criteria were used to establish identity between the in vitro products and the virion proteins: comigration during sodium dodecyl sulfatepolyacrylamide gel electrophoresis, cochromatography in sodium dodecyl sulfate-hydroxyapatite, specific immunoprecipitation of the precursor to core protein VII, and tryptic peptide analysis.