In vitro heterologous cytotoxicity by T effector cells from mice immunized with Sindbis virus.

An in vitro correlate of cell-mediated cross-protection among alpha-viruses was demonstrated by cytotoxicity of Sindbis-immune spleen cells from mice to both Sindbis and Semliki Forest virus (SFV)-infected target cells. This cytotoxicity was shown to be mediated by the T cell population of the spleen and was independent of the presence of macrophages or B cells. The time when the level of the lymphocyte-mediated cytotoxicity (LMC) to SFV-infected cells was maximal coincides with the time when immunity to SFV is maximal in vivo, as reported previously, and when adoptive immunity to SFV can be transferred. After one i.p. injection of Sindbis virus, the level of homologous LMC was higher than the level of heterologous LMC. However, following a second injection of Sindbis virus as immunogen, at a time when the mice are cross-protected to SFV, the heterologous LMC was considerably higher than homologous LMC. We propose that there is suppression of the effector T cells specific for Sindbis-infected cells after the second immunizing injection, probably by homologous antibody. In contrast, there appears to be an anamnestic cell-mediated response to SFV.     

Involvement of cytoplasmic membranes in the non-lytic infection of K-562 cells by Semliki Forest virus

An analysis of the human leukemia cell line, K-562, infected with Semliki Forest virus, has been made with transmission electron microscopy. In contrast to the usual surface budding of the enveloped virus on the plasma membrane of vertebrate cells leading to cytolysis within 20 h, K-562 cells do not show surface budding, and the cells remain intact for periods of several months. Several unusual features of the infection include: 1) the rough endoplasmic reticulum arranges early into continuous perinuclear chains; 2) during the time of virus replication and release, the nucleocapsids aggregate on the cytoplasmic side of internal vesicles in the region of the cell where the Golgi complex is normally located; and 3) during this same time period, the vesicles are seen to contain enveloped virions and rod-like formations, a result suggesting that budding has occurred into these vesicles. Viruses are presumably released from the cell as these vesicles fuse with the plasma membrane. By 12 days post-infection and thereafter, the intact cells show electron-dense aggregates of chromatin, large vacuoles and lipid inclusions throughout the cytoplasm, and only a few virion-containing vesicles.     

Detection of immunologically cross-reacting capsid protein of alphaviruses on the surfaces of infected L929 cells.

Hyperimmune, but not normal immune, monospecific antiserum made to capsid protein of Sindbis virus (SIN) was found to cause cytolysis equally well of both SIN- and Semliki Forest virus-infected L929 cells in antibody-dependent, complement-mediated cytotoxicity assays. The cell surface reactivity of the hyperimmune antiserum was also demonstrated by solid-phase radioimmune assays with unfixed infected cells or infected cells fixed with low concentrations of glutaraldehyde (0.025%) before reactivity with antisera. Higher concentrations of glutaraldehyde lowered the sensitivity of detection. Purified SIN capsid protein specifically inhibited antibody-dependent, complement-mediated cytotoxicity by the monospecific anti-capsid protein serum on SIN- and Semliki Forest virus-infected target cells. That hyperimmune anti-SIN serum also cross-reacts with capsid protein on the surface of Semliki Forest virus-infected cells was suggested by the fact that capsid protein inhibited cross-cytolysis in the antibody-dependent, complement-mediated cytotoxicity assay. The latter antiserum was collected after repeated injections of purified virions over a 9-month period. The results suggest that hyperimmune monospecific antisera made to SIN capsid protein or hyperimmune antisera to SIN or Semliki Forest virions detect homologous and cross-reacting capsid protein determinants on the surface of infected cells.     

Biodeterioration of Mayan buildings at uxmal and tulum,Mexico

Uxmal and Tulum are two important Mayan sites in the Yucatan peninsula. The buildings are mainly composed of limestone and grey/black discoloration is seen on exposed walls and copious greenish biofilms on inner walls. The principal microorganisms detected on interior walls at both Uxmal and Tulum were cyanobacteria; heterotrophic bacteria and filamentous fungi were also present. A dark‐pigmented mitosporic fungus and Bacillus cereus, both isolated from Uxmal, were shown to be acidogenic in laboratory cultures. Cyanobacteria belonging to rock‐degrading genera Synechocystis and Gloeocapsa were identified at both sites. Surface analysis previously showed that calcium ions were present in the biofilms on buildings at Uxmal and Tulum, suggesting the deposition of biosolubilized stone. Apart from their potential to degrade the substrate, the coccoid cyanobacteria supply organic nutrients for bacteria and fungi, which can produce organic acids, further increasing stone degradation.     

Multimodal pressure-flow method to assess dynamics of cerebral autoregulation in stroke and hypertension

Background

This study evaluated the effects of stroke on regulation of cerebral blood flow in response to fluctuations in systemic blood pressure (BP). The autoregulatory dynamics are difficult to assess because of the nonstationarity and nonlinearity of the component signals.

Methods

We studied 15 normotensive, 20 hypertensive and 15 minor stroke subjects (48.0 ± 1.3 years). BP and blood flow velocities (BFV) from middle cerebral arteries (MCA) were measured during the Valsalva maneuver (VM) using transcranial Doppler ultrasound.

Results

A new technique, multimodal pressure-flow analysis (MMPF), was implemented to analyze these short, nonstationary signals. MMPF analysis decomposes complex BP and BFV signals into multiple empirical modes, representing their instantaneous frequency-amplitude modulation. The empirical mode corresponding to the VM BP profile was used to construct the continuous phase diagram and to identify the minimum and maximum values from the residual BP (BP_R) and BFV (BFV_R) signals. The BP-BFV phase shift was calculated as the difference between the phase corresponding to the BP_R and BFV_R minimum (maximum) values. BP-BFV phase shifts were significantly different between groups. In the normotensive group, the BFV_R minimum and maximum preceded the BP_R minimum and maximum, respectively, leading to large positive values of BP-BFV shifts.

Conclusion

In the stroke and hypertensive groups, the resulting BP-BFV phase shift was significantly smaller compared to the normotensive group. A standard autoregulation index did not differentiate the groups. The MMPF method enables evaluation of autoregulatory dynamics based on instantaneous BP-BFV phase analysis. Regulation of BP-BFV dynamics is altered with hypertension and after stroke, rendering blood flow dependent on blood pressure.

     

Further characterization of the binding of human recombinant interleukin 2 to heparin and identification of putative binding sites

We have previously provided compelling evidence that human recombinant interleukin 2 (IL-2) binds to the sulfated polysaccharides heparin, highly sulfated heparan sulfate and fucoidan. Here we show that IL-2 binding is dependent on heparin chain length, but with fragments as small as 15-mers retaining binding activity. The addition of exogenous heparin has no effect on the in vitro biological activity of IL-2. In addition soluble IL-2 receptor alpha and beta polypeptides do not compete with heparin for the binding of IL-2. IL-2 bound by heparin is still recognized by two IL-2 specific monoclonal antibodies, 3H9 and H2- 8, whose epitopes lie in the amino terminal region. Murine IL-2 unlike its human counterpart fails to bind to heparin. Human IL-2 analogs with single amino acid substitutions at positions Lys43, Thr51, and Gln126 analogs no longer bind to heparin. By contrast the Arg38Ala analog retains heparin full heparin binding activity. These experimental findings together with molecular modeling studies suggest two putative heparin binding sites on human IL-2, one involving four basic residues, Lys48, Lys49, Lys54, and His55, and the other being a discontinuous site comprising Lys43, Lys64, Arg81, and Arg83. Neither of these two clusters is completely conserved in murine IL-2. Overall our data suggest that the binding of human IL-2 to heparin and heparan sulfate does not interfere with IL-2/IL-2 receptor interactions. Therefore, binding to glycosaminoglycan may be a mechanism for retaining the cytokine in an active form close to its site of secretion in the tissue, thus favoring a paracrine role for IL-2.      

Efflux pumps expression and its association with porin down-regulation and β-lactamase production among <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> causing bloodstream infections in Brazil

Background  

Multi-drug efflux pumps have been increasingly recognized as a major component of resistance in P. aeruginosa. We have investigated the expression level of efflux systems among clinical isolates of P. aeruginosa, regardless of their antimicrobial susceptibility profile.     

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies with Aspidosperma ramiflorum in vivo and in vitro

Several species of Aspidosperma plants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellow peroba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorum extracts, the plant activity against Plasmodium falciparum was evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium berghei in mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.