Regulation of Abortion by γλ T Cells

PROBLEM: T cells are present at the feto-maternal interface, but their function during pregnancy has not been fully elucidated. T cells bearing γλ T-cell receptor (TCR) may be particularly important, as some subsets can react to trophoblast cells by producing cytokines, such as interleukin-2 (IL-2). METHOD: We depleted T cells bearing the γλ receptor by injecting monoclonal antibodies (mAB) into females of the abortion-prone animal model CBA x DBA/2. We investigated the percentage and number of γλ T-cell receptor positive (TCR)⁺ cells in decidua and spleen during pregnancy in control and γλ-depleted female mice. Pregnant females were also exposed to ultrasonic sound stress to boost the abortion rate. RESULTS: Stress failed to increase the abortion rate in the γλ TCR-depleted mice. FACScan analysis show that the ratio of cells bearing the γλ TCR dramatically decreased after injection of mAB to the γλ TCR in spleen and decidua, these cells recovered six days after depletion, showing a change in cytokine pattern. Levels of TNF-α in decidual γλ T cells decreased; similar effects of decreasing Th₁ cytokines could be observed in splenic γλ T cells. We further identified increased levels of intracellular TNF-α in the Vλ4 subset in the decidua, compared to spleen. CONCLUSIONS: Trophoblast recognition by the Vλ4 T-cell subset in the decidua may cause the release of abortogenic cytokines such as TNF-α. Depletion of such γλ TCR T cells during early pregnancy may promote successful pregnancy outcome in normal pregnancy and prevent stress-induced abortions.     

Development and activation of human dendritic cells in vivo in a xenograft model of human hematopoiesis

Dendritic cells (DCs) are derived from CD34+ progenitors and play a central role in the development of immune responses and in tolerance. Their therapeutic potential underscores the need for in vivo models that accurately recapitulate human DC development and function to provide a better understanding of DC biology in health and disease. Using nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice transplanted with human CD34+ cells as a model of human hematopoiesis, we examined DC ontogeny. Progenitors of both myeloid (m) and plasmacytoid (p) DCs were identified in the bone marrow of mice up to 24 weeks after transplant, indicating ongoing and sustained production of DCs after initial engraftment. To determine whether human DCs derived from transplanted stem cells were functional, their response to acute inflammation using lipopolysaccharide (LPS) was examined. Eighteen hours after LPS administration, a dramatic increase in the plasma levels of the human inflammatory cytokines interleukin (IL)-8, IL-10, tumor necrosis factor-alpha, and IL-12p70 was observed. Only mDCs and not pDCs responded in vivo to LPS by upregulating CD86 and CD83. In vivo activation of human mDCs resulted in a substantial increase in the ability of mDCs to induce the proliferation of naive human T cells. Taken together, these data indicate that human CD34+ cells seem to have differentiated appropriately within the NOD/SCID microenvironment into DCs that are developmentally, phenotypically, and functionally similar to the DC subsets found in humans.     

Tularemia

Francisella tularensis is the etiological agent of tularemia, a serious and occasionally fatal disease of humans and animals. In humans, ulceroglandular tularemia is the most common form of the disease and is usually a consequence of a bite from an arthropod vector which has previously fed on an infected animal. The pneumonic form of the disease occurs rarely but is the likely form of the disease should this bacterium be used as a bioterrorism agent. The diagnosis of disease is not straightforward. F. tularensis is difficult to culture, and the handling of this bacterium poses a significant risk of infection to laboratory personnel. Enzyme-linked immunosorbent assay- and PCR-based methods have been used to detect bacteria in clinical samples, but these methods have not been adequately evaluated for the diagnosis of pneumonic tularemia. Little is known about the virulence mechanisms of F. tularensis, though there is a large body of evidence indicating that it is an intracellular pathogen, surviving mainly in macrophages. An unlicensed live attenuated vaccine is available, which does appear to offer protection against ulceroglandular and pneumonic tularemia. Although an improved vaccine against tularemia is highly desirable, attempts to devise such a vaccine have been limited by the inability to construct defined allelic replacement mutants and by the lack of information on the mechanisms of virulence of F. tularensis. In the absence of a licensed vaccine, aminoglycoside antibiotics play a key role in the prevention and treatment of tularemia.     

Antigenic properties of HpaA and Omp18, two outer membrane proteins of Helicobacter pylori

Outer membrane proteins (OMPs) are incorporated into the outer plasma membrane of Helicobacter pylori and are important for, e.g., ion transport, adherence, structural and osmotic stability, and bacterial virulence but may also be antigenic due to their surface exposure. Previous proteome-based approaches with H. pylori lysates determined a strong serological reaction towards two H. pylori OMPs, HpaA (TIGR HP0797) and Omp18 (TIGR HP1125). PCR was used to detect DNA encoding the two proteins, and a positive signal was found in all H. pylori strains tested. Proteins were cloned and expressed in the human kidney cell line HK293 with the QiaExpressionist system with a C-terminal His tag. Only sera from infected persons showed a positive reaction with the recombinant proteins. Recombinant HpaA (rHpaA) and rOmp18 were incubated with human peripheral blood mononuclear cells and induced secretion of interleukin-12 (IL-12) and IL-10 from these cells. To determine the effect on antigen-presenting cells, human blood monocytic and dendritic cells (DCs) were isolated by magnetic cell separation. rOmp18 and rHpaA strongly stimulated major histocompatibility class II and CD83 expression 7- to 10-fold on isolated DCs. rHpaA and rOmp18 failed to stimulate IL-8 secretion from monocytes but increased secretion of IL-12 and IL-10 from DCs significantly. In summary, HpaA and Omp18 are recognized by human dendritic cells and induce their maturation as well as antigen presentation. HpaA and Omp18 of H. pylori thereby appear to have a specific antigenic potential in humans.     

Identification of two phylogenetically related organisms from feces by PCR for detection of Salmonella spp

Two previously reported PCR methods were evaluated to determine whether they are as sensitive and specific as conventional culture methods in detecting Salmonella spp. from feces. Bovine and equine feces were enriched overnight in brain heart infusion broth and assayed using PCR methods and primer sets described by other investigators. A total of 774 fecal specimens were tested using a primer set (invE-A primer set) that amplifies a region spanning the invasin E and A genes of Salmonella enterica serovar Typhimurium. A subset of these fecal specimens (306 of the 774 total) were tested using primers (hisJ primer set) that amplify a portion of the histidine transport J gene. The PCR required 24 h to obtain results, whereas it took 5 to 7 days to identify Salmonella spp. by culture. PCR detection of Salmonella spp. using the hisJ primers and the invE-A primers had a sensitivity of 93.3 and 80%, respectively, and a specificity of 85.6 and 98.6%, respectively, compared with bacterial culture. Amplification of 42 culture-negative fecal specimens (of 306 total specimens) generated a DNA fragment that corresponded to the molecular weight of the amplified hisJ gene. The hisJ-generated amplicons from six culture-negative and six culture-positive specimens were sequenced and analyzed using DNA sequence alignment and phylogenetic analysis software. A neighbor-joining dendrogram of the DNA sequences of both sets of hisJ amplicons revealed two distinct groups-one group of amplicons from culture-positive specimens identical to the hisJ gene of S. enterica serovar Typhimurium and a second group of amplicons from culture-negative specimens that were more closely related to hisJ of S. enterica serovar Typhimurium than to other hisJ sequences present in nucleotide databases.     

Detection and identification of fungi from fungus balls of the maxillary sinus by molecular techniques

The aim of this study was to find a reliable method for the detection and identification of fungi in fungus balls of the maxillary sinus and to evaluate the spectrum of fungi in these samples. One hundred twelve samples were obtained from patients with histologically proven fungal infections; 81 samples were paraffin-embedded tissue sections of the maxillary sinus. In 31 cases, sinus contents without paraffin embedding were sent for investigation. PCR amplification with universal fungal primers for 28S ribosomal DNA and amplicon identification by hybridization with species-specific probes for Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus glaucus, Pseudallescheria boydii, Candida albicans, and Candida glabrata were performed for all samples. Furthermore, PCR products were sequenced. Fresh samples were also cultivated. Fungal DNA was detected in all of the fresh samples but only in 71 paraffin-embedded tissue samples (87.7%). Sequence analysis was the most sensitive technique, as results could be obtained for 28 (90.3%) fresh samples by this method in comparison to 24 (77.4%) samples by hybridization and 16 (51.6%) samples by culture. However, sequence analysis delivered a result for only 36 (50.7%) of the paraffin-embedded specimens. Hybridization showed reliable results for A. fumigatus, which proved to be the most common agent in fungus balls of the maxillary sinus. Other Aspergillus species and other genera were rarely found.     

Mechanical Activation of Pharmaceutical Systems

Mechanical activation has become a phenomenon of general significance in pharmaceutics. This report describes the extent of activation induced by relevant processes. With the use of the Eyring equation the transformation of structurally stored energy into chemical energy and the implied free enthalpy as well as the excess free enthalpy (activity) were evaluated from the rate of an indicator reaction. A comparison was made between different kinds of milling and tabletting with respect to pharmaceutical conditions. An optimization of these processes was derived.     

Importance of Structural Free Space to the Solvent Power of Water

The contribution of structural free space to the solvent power of water was examined by a systematic modification of the geometric factor. Gaps and holes, available to foreign molecule occupation, are thought to be filled at low concentrations (max. 1 %) of aliphatic alcohols. The effect upon solvency reached approximately 10%, which suggests that spatial parameters affect solvent power. The results demonstrate the importance of solvent purity in the dissolution process.     

Krill Oil Supplementation Reduces Exacerbated Hepatic Steatosis Induced by Thermoneutral Housing in Mice with Diet-Induced Obesity

Preclinical evidence suggests that n-3 fatty acids EPA and DHA (Omega-3) supplemented as phospholipids (PLs) may be more effective than triacylglycerols (TAGs) in reducing hepatic steatosis. To further test the ability of Omega-3 PLs to alleviate liver steatosis, we used a model of exacerbated non-alcoholic fatty liver disease based on high-fat feeding at thermoneutral temperature. Male C57BL/6N mice were fed for 24 weeks a lard-based diet given either alone (LHF) or supplemented with Omega-3 (30 mg/g diet) as PLs (krill oil; ω3PL) or TAGs (Epax 3000TG concentrate; ω3TG), which had a similar total content of EPA and DHA and their ratio. Substantial levels of TAG accumulation (~250 mg/g) but relatively low inflammation/fibrosis levels were achieved in the livers of control LHF mice. Liver steatosis was reduced by >40% in the ω3PL but not ω3TG group, and plasma ALT levels were markedly reduced (by 68%) in ω3PL mice as well. Krill oil administration also improved hepatic insulin sensitivity, and its effects were associated with high plasma adiponectin levels (150% of LHF mice) along with superior bioavailability of EPA, increased content of alkaloids stachydrine and trigonelline, suppression of lipogenic gene expression, and decreased diacylglycerol levels in the liver. This study reveals that in addition to Omega-3 PLs, other constituents of krill oil, such as alkaloids, may contribute to its strong antisteatotic effects in the liver.     

Active Case Finding of Current Bornavirus Infections in Human Encephalitis Cases of Unknown Etiology,Germany, 2018–2020

Human bornavirus encephalitis is a severe and often fatal infection caused by variegated squirrel bornavirus 1 (VSBV-1) and Borna disease virus 1 (BoDV-1). We conducted a prospective study of bornavirus etiology of encephalitis cases in Germany during 2018–2020 by using a serologic testing scheme applied along proposed graded case definitions for VSBV-1, BoDV-1, and unspecified bornavirus encephalitis. Of 103 encephalitis cases of unknown etiology, 4 bornavirus infections were detected serologically. One chronic case was caused by VSBV-1 after occupational-related contact of a person with exotic squirrels, and 3 acute cases were caused by BoDV-1 in virus-endemic areas. All 4 case-patients died. Bornavirus etiology could be confirmed by molecular methods. Serologic testing for these cases was virus specific, discriminatory, and a practical diagnostic option for living patients if no brain tissue samples are available. This testing should be guided by clinical and epidemiologic suspicions, such as residence in virus-endemic areas and animal exposure.