Members of the heat-shock protein 70 family promote cancer cell growth by distinct mechanisms

Whereas the stress-inducible heat-shock protein 70 (Hsp70) has gained plenty of attention as a putative target for tumor therapy, little is known about the role of other Hsp70 proteins in cancer. Here we present the first thorough analysis of the expression and function of the cytosolic Hsp70 proteins in human cancer cells and identify Hsp70-2, a protein essential for spermatogenesis, as an important regulator of cancer cell growth. Targeted knock-down of the individual family members by RNA interference revealed that both Hsp70 and Hsp70-2 were required for cancer cell growth, whereas the survival of tumorigenic as well as nontumorigenic cells depended on Hsc70. Cancer cells depleted for Hsp70 and Hsp70-2 displayed strikingly different morphologies (detached and round vs. flat senescent-like), cell cycle distributions (G2/M vs. G1 arrest) and gene expression profiles. Only Hsp70-2 depletion induced the expression of macrophage inhibitory cytokine-1 that was identified as a target of P53 tumor-suppressor protein and a mediator of the G1 arrest and the senescent phenotype. Importantly, concomitant depletion of Hsp70 and Hsp70-2 had a synergistic antiproliferative effect on cancer cells. Thus, highly homologous Hsp70 proteins bring about nonoverlapping functions essential for cell growth and survival.     

Comparison of two urinary antigen tests for establishment of pneumococcal etiology of adult community-acquired pneumonia

The Binax NOW immunochromatographic test (ICT) detecting the pneumococcal C polysaccharide and a serotype-specific latex agglutination (LA) test detecting 23 pneumococcal capsular antigens were evaluated for establishing pneumococcal etiology in community-acquired pneumonia (CAP) by use of nonconcentrated urine. ICT was considered to be strongly positive for result lines at least as intense as the control line and weakly positive for less intense result lines. When 215 adult CAP patients were tested, strong ICT, weak ICT, and LA positivity were found in 28, 24, and 16 patients, respectively; of these patients, 13 (46%), 6 (25%), and 13 (81%), respectively, had pneumococcal bacteremia and 27 (96%), 17 (71%), and 15 (94%), respectively, had Streptococcus pneumoniae isolated from blood, sputum, and/or nasopharynx. Among 108 controls tested, 2 (1.9%) were weakly ICT positive. When weak positivity was considered negative, the sensitivity of ICT decreased from 79% (19 of 24) to 54% (13 of 24), while the specificity increased from 83% (158 of 191) to 92% (176 of 191); no controls were false positive. The sensitivity and specificity of LA were 54% (13 of 24) and 98% (188 of 191), respectively. Eight of nine LA serotypes corresponded to culture serotypes. In conclusion, using nonconcentrated urine and dividing ICT-positive results into strongly and weakly positive results is a suitable way of performing ICT. While weak ICT positivity should be interpreted with caution, strong ICT positivity and LA positivity should be considered supportive of pneumococcal etiology in adult CAP. As such, these assays might have implications for antibiotic use in CAP. LA has promising potential for pneumococcal serotyping, although further evaluation is required.     

CXCR4 and CCR5 expression on CD4+ T cells in vivo and HIV-1 antigen beta-chemokine production in vitro after treatment with HIV-1 immunogen (REMUNE)

BACKGROUND: The chemokine receptors CXCR4 and CCR5 have been identified as the major coreceptors for HIV-1 on CD4+ cells and macrophages. The natural ligands for these receptors are SDF-1 and the beta-chemokines (MIP-1alpha, MIP-1beta, RANTES), respectively, and are the products of a variety of immune cells, including CD8+ T lymphocytes. STUDY DESIGN/METHODS: We hypothesized that the ability to stimulate the natural ligands for these receptors using an immune based therapy might influence in vivo chemokine receptor expression. RESULTS: In vivo CXCR4 expression remained stable after treatment with an HIV-1 Immunogen (REMUNE), whereas CCR5 expression on CD4+ T cells decreased (p < .05). Furthermore, HIV-1 antigen-specific production of beta-chemokines in vitro was also augmented (P < .05). CONCLUSIONS: These preliminary results suggest that this HIV-1-specific immune-based therapy can stimulate antigen-specific beta-chemokine production in vitro and downregulate CCR5 receptor expression on CD4 cells in vivo.     

Expansion of CD5 - B cells in multiple sclerosis correlates with CD80 (B7-1) expression

The pathogenetic role of autoantibodies in multiple sclerosis (MS) is uncertain. CD5+ B cells commonly produce autoantibodies, but CD5 expression has also been implicated in B-cell tolerance. We studied B-cell subsets, anti-myelin protein antibody-secreting cells in cerebrospinal fluid (CSF) and a panel of serum autoantibodies in patients with clinically isolated syndromes (CIS), suggestive of MS and patients with clinically definite MS (CDMS). Patients with CDMS had a higher percentage of CD5- B cells in CSF than did control subjects (P = 0.02). CIS patients with immunoglobulin G (IgG) oligoclonal bands in CSF or multiple lesions on magnetic resonance imaging (MRI) had a higher percentage of CD5- B cells in CSF than did the remaining CIS patients (P = 0.03). The percentage of CD5- and CD80+ B cells correlated positively and the percentage of CD5+ B cells correlated negatively with the number of CSF cells secreting anti-myelin basic protein (anti-MBP) antibodies. The prevalence of serum autoantibodies was comparable in the three patient groups. We conclude that intrathecal expansion of CD5- B cells appears to be more characteristic in MS patients, and CD5+ B cells may be associated with a lower prevalence of anti-myelin antibody production.     

Faster activation of polymorphonuclear neutrophils in resistant mice during early innate response to Pseudomonas aeruginosa lung infection

Polymorphonuclear neutrophils (PMNs) are crucial for the outcome of Pseudomonas aeruginosa lung infection in patients with cystic fibrosis. We compared PMNs and inflammatory cytokines in the lungs and blood from susceptible BALB/c and resistant C3H/HeN mice 1 and 2 days after intratracheal challenge with alginate embedded P. aeruginosa. These parameters were correlated with the quantitative bacteriology and histopathology of the lungs. After challenge, the content of granulocyte colony-stimulating factor (G-CSF) and macrophage inflammatory protein-2 (MIP-2) was increased in the lungs and the sera and the percentage of PMNs was increased in the blood. However, 2 days after challenge the concentration of G-CSF and MIP-2 was higher in the lungs and sera of BALB/c mice. CD11b expression was higher on the PMNs of the C3H/HeN mice. The expression of CD62L on PMNs of both strains of mice was decreased 1 day after bacterial challenge, whereas the expression was increased after 2 days of challenge on PMNs of C3H/HeN mice only. These changes were accompanied by a more severe lung inflammation in BALB/c mice and faster clearance of the bacteria in C3H/HeN mice. In conclusion, the rapid early bacterial clearance in the lungs of C3H/HeN mice could be explained by faster activation of the PMNs, as indicated by the higher up-regulation of CD11b. The severe lung inflammation in BALB/c mice may be caused by the early higher content of G-CSF in the sera mobilizing PMNs from the bone marrow and the persistent chemotactic gradient provided by MIP-2 in the lungs.     

Improved outcome of chronic Pseudomonas aeruginosa lung infection is associated with induction of a Th1-dominated cytokine response

Repeated challenge with antigen is involved in the pathogenesis of a variety of pulmonary diseases. Patients with cystic fibrosis (CF) experience recurrent pulmonary colonization with Pseudomonas aeruginosa before establishment of chronic lung infection. To mimic recurrent lung infections in CF patients, the lungs of susceptible BALB/c mice were re-infected with P. aeruginosa 14 days after the initial infection. Singly-infected BALB/c mice, as well as non-infected mice, were used as controls. Decreased mortality and milder lung inflammation in re-infected BALB/c mice, as well as a tendency for improved clearance of bacteria, was observed when compared with singly-infected mice. The improved outcome in re-infected mice correlated with changes in CD4 cell numbers. Surface expression of LFA-1 on pulmonary CD4 cells was increased in re-infected compared with singly-infected mice. Moreover, resistance to re-infection was paralleled by a shift towards a Th1-dominated response and increased IL-12 production. No significant increase in serum IgG was observed in the re-infected mice. In conclusion, these results indicate a protective role for a Th1-dominated response, independent of antibody production, in chronic P. aeruginosa lung infection in CF.     

Gentamicin susceptibility in Escherichia coli related to the genetic background: problems with breakpoints

In total, 120 Escherichia coli isolates positive for one of the gentamicin resistance (GEN(R)) genes aac(3)-II, aac(3)-IV or ant(2')-I were tested for gentamicin susceptibility by the agar dilution method. Isolates positive for aac(3)-IV or ant(2')-I had an MIC distribution of 8-64 mg/L, whereas isolates positive for aac(3)-II had MICs of 32 to >512 mg/L, suggesting a relationship between the distribution of MICs and the specific GEN(R) mechanism. The MIC distribution, regardless of the GEN(R) mechanism, was 8 - >512 mg/L, which supports the clinical breakpoint of MIC >4 mg/L suggested by EUCAST and questions the breakpoint recommended by the CLSI (> or =16 mg/L).     

Motor unit recruitment and rate coding in response to fatiguing shoulder abductions and subsequent recovery

The purpose of the present study was to investigate motor unit (MU) recruitment and firing rate, and the MU action potential (MUAP) characteristics of the human supraspinatus muscle during prolonged static contraction and subsequent recovery. Eight female subjects sustained a 30° shoulder abduction, requiring 11–12% of maximal voluntary contraction (MVC), for 30 min. At 10 and 30 min into the recovery period, the shoulder abduction was repeated for 1 min. The rating of perceived exertion for the shoulder region increased to “close to exhaustion” during the prolonged contraction, and the surface electromyography (EMG) recorded from the deltoid and trapezius muscles showed signs of local muscle fatigue. From the supraspinatus muscle, a total of 23,830 MU firings from 265 MUs were identified using needle electrodes. Of the identified MUs, 95% were continuously active during the 8-s recordings, indicating a low degree of MU rotation. The mean (range) MU firing rate was 11.2 (5.7–14.5) Hz, indicating the relative force contribution of individual MUs to be larger than the overall mean shoulder muscle load. The average MU firing rate remained stable throughout the prolonged abduction, although firing rate variability increased in response to fatigue. The average concentric MUAP amplitude increased by 38% from the beginning (0–6 min) to the end (24–29 min) of the contraction period, indicating recruitment of larger MUs in response to fatigue. In contrast, after 10 min of recovery the average MU amplitude was smaller than seen initially in the prolonged contraction, but not different after 30 min, while the MU firing rate was higher during both tests. In conclusion, MU recruitment plays a significant role during fatigue, whereas rate coding has a major priority during recovery. Furthermore, a low degree of MU rotation in combination with a high relative load at the MU level may imply a risk of overloading certain MUs during prolonged contractions. Accepted: 6 June 2000     

Identification of recurrent and novel mutations in the LDL receptor gene in Spanish patients with familial hypercholesterolemia. Mutations in brief no. 135. Online

We used the single strand conformation polymorphism (SSCP) method to investigate 13 apparently unrelated Spanish patients with familial hypercholesterolemia (FH) for mutations in the promoter region and the 18 exons and their flanking intron sequences of the low density lipoprotein (LDL) receptor gene. We found 16 aberrant SSCP patterns, and the underlying mutations were characterized by DNA sequencing. Five novel missense mutations, Q71E, C74G, C95R, C281Y and D679E, and one nonsense mutation, Q133X, were identified. We also found six missense mutations, S156L, D200Y, D200G, E256K, T413K and C646Y, and one stop codon mutation, W(-18)X, that were previously described in patients from other populations. A new frameshift mutation, 2085del19, was found in one patient. We also identified three splicing mutations; two of them are novel mutations, 1706-10G->A and 2390-1G->A, and the other one has been reported recently, 313+1G->C. Four patients were found to carry two different mutations in the same allele: Q71E and 313+1G->C; C95R and D679E; W(-18)X and E256K, and C281Y and 1706-10G->A. Our results demonstrate that there is a broad spectrum of mutations in the LDL receptor gene in the Spanish population.     

Subpopulations of T lymphocytes and non-specific suppressor cell activity in patients with atopic dermatitis.

Thirty-five adult persons with moderate to severe atopic dermatitis and increased levels of IgE in serum were studied for the presence of Fc-IgG (T gamma) and Fc-IgM (T mu) receptor-carrying T lymphocytes in peripheral blood. When T lymphocytes from the patient were kept in vitro cultures a reduced expansion of the Fc-IgG receptors was found after 24 hr of incubation, but not after 48 hr of incubation. No difference was observed concerning Fc-IgM-carrying T lymphocytes. In 19 patients we studied Con A-induced suppressor activity of blood lymphocytes using autologous PHA-stimulated lymphocytes as target cells. Following stimulation with Con A, 1 microgram/ml, for 2 and 4 days, lymphocytes from atopic patients had a reduced non-specific suppressor activity in vitro. If however, the concentration of Con A was increased to 10 microgram/ml, then lymphocytes from atopic patients exhibited a normal suppressor activity.