Central GABAA and GABAB receptor modulation of basal and stress-induced plasma interleukin-6 levels in mice

The development of control strategies for loiasis is of crucial importance in endemic areas and depends heavily on the accurate identification of occult-infected individuals. A polymerase chain reaction (PCR) and nested polymerase chain reaction (nested PCR) were developed and based on sequences of the repeat 3 region (15r3) of the gene encoding a Loa loa 15-kD protein. The assays was performed on 20 blood samples from occult-infected subjects and 30 from field-collected amicrofilaremic individuals. The size of the initial PCR product was 396 basepairs (bp). When this initial amplification using primers 15r3(1) and 15r3(2) was carried out for 30 cycles, the PCR products from three of the 20 occult-infected and five of the 30 amicrofilaremic individuals were visualized after electrophoresis by staining the gel with ethidium bromide. Subsequent Southern blotting and hybridization with the specific probe revealed hybridization in 19 of 20 occult-infected and 23 of 30 amicrofilaremic samples but only after two days of exposure of the blot to the x-ray film. When the nested PCR was carried out (product size = 366 bp, primers 15r3(3) and 15r3(4)), 19 of 20 occult-infected and 23 of 30 amicrofilaremic samples that were positive by Southern hybridization of the initial PCR products were strongly positive by staining with ethidium bromide. Qualitative Southern blotting of the nested PCR products using the same probe previously described confirmed the ethidium bromide staining results after a very short exposure time of 4 hr. These results demonstrate that the nested PCR amplification product is specific and that its sensitivity in detecting occult loiasis is 95%. This approach has significant promise for the screening of large human populations for active loiasis without the requirement for blotting and hybridization of the PCR products.     

Class sigma glutathione transferase unfolds via a dimeric and a monomeric intermediate: impact of subunit interface on conformational stability in the superfamily

Solvent-induced equilibrium unfolding of a homodimeric class sigma glutathione transferase (GSTS1-1, EC 2.5.1.18) was characterized by tryptophan fluorescence, anisotropy, enzyme activity, 8-anilino-1-naphthalenesulfonate (ANS) binding, and circular dichroism. Urea induces a triphasic unfolding transition with evidence for two well-populated thermodynamically stable intermediate states of GSTS1-1. The first unfolding transition is protein concentration independent and involves a change in the subunit tertiary structure yielding a partially active dimeric intermediate (i.e., N2 left and right arrow I2). This is followed by a protein concentration dependent step in which I2 dissociates into compact inactive monomers (M) displaying enhanced hydrophobicity. The third unfolding transition, which is protein concentration independent, involves the complete unfolding of the monomeric state. Increasing NaCl concentrations destabilize N2 and appear to shift the equilibrium toward I2 whereas the stability of the monomeric intermediate M is enhanced. The binding of substrate or product analogue (i.e., glutathione or S-hexylglutathione) to the protein's active site stabilizes the native dimeric state (N2), causing the first two unfolding transitions to shift toward higher urea concentrations. The stability of M was not affected. The data implicate a region at/near the active site in domain I (most likely alpha-helix 2) as being highly unstable/flexible which undergoes local unfolding, resulting initially in I2 formation followed by a disruption in quaternary structure to a monomeric intermediate. The unfolding/refolding pathway is compared with those observed for other cytosolic GSTs and discussed in light of the different structural features at the subunit interfaces, as well as the evolutionary selection of this GST as a lens crystallin.     

Mast cell activation and migration to lymph nodes during induction of an immune response in mice

The mast cell response in skin and lymph nodes was examined during the sensitization phase of dinitrofluorobenzene (DNFB)-induced contact hypersensitivity in mice. Degranulation of 62% of mast cells in DNFB-exposed skin was evident within 30 min of a dual application of DNFB, reaching a peak of 77% at 24 h, and persisting in 42% after 5 d. Abundant expression of macrophage inflammatory protein (MIP)-1alpha and MIP-1beta mRNAs and proteins was observed in keratinocytes, and mast cell degranulation was significantly inhibited after administration of neutralizing antibodies to MIP-1alpha, but not MIP-1beta. During DNFB sensitization, the mast cell density in the skin decreased by half, concurrent with a fivefold expansion of mast cell numbers in draining lymph nodes. Fluorescent-labeled mast cells injected into the skin appeared in draining lymph nodes after application of DNFB, followed by subsequent migration to the spleen. In lymph nodes, mast cells were an abundant and predominant source of MIP-1beta, neutralization of which partially inhibited T lymphocyte recruitment. These results indicate that mast cells contribute to the induction of this primary immune response by activation at and migration from the site of antigen encounter to draining lymph nodes, wherein they mediate T lymphocyte recruitment by production of MIP-1beta.     

Comparison of the resistance of C57BL/6 and C3H/He mice to infection with Mycobacterium paratuberculosis

Susceptibility of C57BL/6 (Bcgs) and C3H/HeN (Bcgr) mice to an intraperitoneal infection with Mycobacterium paratuberculosis strain 19698 was compared (by histopathology and the number of mycobacteria isolated from the spleen). Mycobacterial counts from the spleen of Bcgr mice progressively decreased over the course of infection but remained unchanged in Bcgs mice. Granulomatous lesions and acid-fast bacteria were consistently present in the liver and lymph nodes of Bcgs mice, whereas lesions were transient or absent in Bcgr mice. These results indicate that Bcgr mice are inherently resistant to M. paratuberculosis, whereas Bcgs mice are inherently susceptible. These differences may prove useful in elucidating the mechanisms of resistance and susceptibility to paratuberculosis and other mycobacterial infections.     

Physical fitness, physical activity, and functional limitation in adults aged 40 and older

PURPOSE: A cohort of middle-aged and older men and women were followed for an average of 5.5 yr to examine the association between physical fitness, physical activity, and the prevalence of functional limitation. METHODS: The participants received medical assessments between 1980 and 1988 and responded to a mail-back survey regarding functional status in 1990. RESULTS: Among 3495 men and 1175 women over 40 yr of age at baseline, 350 (7.5%) reported at least one functional limitation in daily or household activities at follow-up. The prevalence of functional limitation was higher among women than men. Physically fit and physically active participants reported less functional limitation than unfit or sedentary participants. After controlling for age and other risk factors, the prevalence of functional limitation was lower for both moderately fit (odds ratio = 0.4, 95% CI = 0.2-0.6) and high fit men (odds ratio = 0.3, 95% CI = 0.2-0.4), compared with low fit men. Corresponding figures for women were 0.5 (0.3-0.7) and 0.3 (0.2-0.5) for moderately fit and high fit women. The association between physical activity and functional limitation was similar to the data for physical fitness. CONCLUSIONS: These data support a protective effect of physical fitness and physical activity on functional limitation among older adults and extend this protective effect to middle-aged men and women.     

Disposition and elimination of meropenem in cerebrospinal fluid of hydrocephalic patients with external ventriculostomy

The broad antibacterial spectrum and the low incidence of seizures in meropenem-treated patients qualifies meropenem for therapy of bacterial meningitis. The present study evaluates concentrations in ventricular cerebrospinal fluid (CSF) in the absence of pronounced meningeal inflammation. Patients with occlusive hydrocephalus caused by cerebrovascular diseases, who had undergone external ventriculostomy (n = 10, age range 48 to 75 years), received 2 g of meropenem intravenously over 30 min. Serum and CSF were drawn repeatedly and analyzed by liquid chromatography-mass spectroscopy. Pharmacokinetics were determined by noncompartmental analysis. Maximum concentrations in serum were 84.7 +/- 23.7 microg/ml. A CSF maximum (CmaxCSF) of 0.63 +/- 0.50 microg/ml (mean +/- standard deviation) was observed 4.1 +/- 2.6 h after the end of the infusion. CmaxCSF and the area under the curve for CSF (AUCCSF) depended on the AUC for serum (AUCS), the CSF-to-serum albumin ratio, and the CSF leukocyte count. Elimination from CSF was considerably slower than from serum (half-life at beta phase [t1/2beta] of 7.36 +/- 2.89 h in CSF versus t1/2beta of 1.69 +/- 0.60 h in serum). The AUCCSF/AUCS ratio for meropenem, as a measure of overall CSF penetration, was 0.047 +/- 0.022. The AUCCSF/AUCS ratio for meropenem was similar to that for other beta-lactam antibiotics with a low binding to serum proteins. The concentration maxima of meropenem in ventricular CSF observed in this study are high enough to kill fully susceptible pathogens. They may not be sufficient to kill bacteria with a reduced sensitivity to carbapenems, although clinical success has been reported for patients with meningitis caused by penicillin-resistant pneumococci and Pseudomonas aeruginosa.     

Human immunodeficiency virus infection, Part II

The acceptance of highly active antiretroviral therapy (HAART) among patients and health care providers has had a dramatic impact on the epidemiology and clinical characteristics of many opportunistic infections associated with human immunodeficiency virus (HIV). Previously intractable opportunistic infections and syndromes are now far less common. In addition, effective antibiotic prophylactic therapies have had a profound impact on the risk of patients developing particular infections and on the incidence of these infections overall. Most notable among these are Pneumocystis carinii, disseminated Mycobacterium avium complex, tuberculosis, and toxoplasmosis. Nevertheless, infections continue to cause significant morbidity and mortality among patients who are infected with HIV. The role of HAART in many clinical situations is unquestioned. Compelling data from clinical trials support the use of these therapies during pregnancy to prevent perinatal transmission of HIV. HAART is also recommended for health care workers who have had a "significant" exposure to the blood of an HIV-infected patient. Both of these situations are discussed in detail in this article. In addition, although more controversial, increasing evidence supports the use of HAART during the acute HIV seroconversion syndrome. An "immune reconstitution syndrome" has been newly described for patients in the early phases of treatment with HAART who develop tuberculosis, M avium complex, and cytomegalovirus disease. Accumulating data support the use of hydroxyurea, an agent with a long history in the field of myeloproliferative disorders, for the treatment of HIV. Newer agents, particularly abacavir and adefovir dipivoxil, are available through expanded access protocols, and their roles are being defined and clarified.     

Reduction of disulfide bonds in an amyloidogenic Bence Jones protein leads to formation of "amyloid-like" fibrils in vitro

Motivated by the finding that the amino acid sequence of the Bence Jones protein BJP-DIA was identical to that of the main protein component of the amyloid fibrils obtained from the same patient with AL-amyloidosis, (Klafki, H.-W., Kratzin, H.-D., Pick, A.-I., Eckart, K., Karas, M. & Hilschmann, N. (1992) Biochemistry 31, 3265-3272.), we attempted to create "amyloid-like" fibrils from the Bence Jones protein in vitro, without addition of proteolytic enzymes. Reduction of BJP-DIA, solubilized in PBS, pH 7.4, overnight at 37 degrees C resulted in the formation of a precipitate which had affinity for the dye Congo red. Electron microscopy of negatively stained samples of the reduced protein revealed aggregates of linear unbranched fibrils. SDS-polyacrylamide gel electrophoresis demonstrated that the precipitate consisted almost exclusively of intact light chain molecules. This result makes it possible to deduce a molecular model of these amyloid fibrils generated in vitro.     

MVA-vaccine in primovaccination against smallpox after the age of three (author's transl)

These are the results of 417 primary vaccinations against smallpox with MVA, an attenuated vaccine. All of the patients were over three years old. In Europe, this group has an increased risk of postvaccinal encephalitis. The intracutaneous inoculation produced a light local infiltration and only 11.7% showed a light generalized reaction. In order to attain a reliable immunity, a second inoculation was performed one week later, epicutaneously with Elstree-vaccine. The local reaction was in general that of a normal revaccination, and 79.4% of our patients had neither fever nor general symptoms. Among the others no one became seriously ill. We did not observe any complications specific for a smallpox vaccination. In comparison to the other vaccination methods currently used, the MVA "Two-step"-vaccination is probably the safest. Its risk of a postvaccinal encephalitis is supposed to be lower according to animal experiments and theoretical considerations. This hypothesis has been confirmed meanwhile by the results of several thousand vaccinations in the already mentioned age-group.