Tetanus toxin fragment C expressed in live Salmonella vaccines enhances antibody responses to its fusion partner Schistosoma haematobium glutathione S-transferase

Tetanus toxoid has been used widely as an adjuvant. The atoxic fragment C from tetanus toxin (TetC) is potently immunogenic when expressed in Salmonella vaccine strains and has been used as a fusion partner for antigens (Ag). However, there has been no formal comparison of the immunomodulatory impact of TetC on its fusion partners. In this study, we have addressed this important issue. The protective 28-kDa glutathione S-transferase (GST) from Schistosoma haematobium (Sh28GST) was expressed either as a fusion to TetC or as the full-length Sh28GST alone in a nonvirulent aroA-attenuated strain of Salmonella enterica serovar Typhimurium. The Sh28GST proteins were soluble and stably expressed in Salmonella, as evaluated by Western blotting with TetC and/or Sh28GST antisera. Mice were immunized orally with a single dose of the live recombinant Salmonella. The constructs were stable in mice but, dramatically, only the strain expressing the TetC-Sh28GST fusion elicited significant antibody (Ab) responses directed against Sh28GST as determined by enzyme-linked immunosorbent assay. An analysis of the isotype profiles showed that these mice also produced anti-Sh28GST immunoglobulin A and GST-neutralizing assays revealed high levels of neutralizing Abs in sera. These are important correlates of protection in schistosomiasis. In addition, stimulation of spleen cells from immunized mice with Sh28GST Ag showed that both strains, expressing Sh28GST alone or the TetC-Sh28GST fusion, were able to stimulate the secretion of Th1-related cytokines (gamma interferon and interleukin 2) to comparable levels. Thus, TetC has modulated the immune responses generated against its fusion partner, Sh28GST, by markedly enhancing the Ab responses elicited. These results have important implications in the rational development of live vaccines.     

Consensus sequence-based scheme for epidemiological typing of clinical and environmental isolates of Legionella pneumophila

A previously described sequence-based epidemiological typing method for clinical and environmental isolates of Legionella pneumophila serogroup 1 was extended by the investigation of three additional gene targets and modification of one of the previous targets. Excellent typeability, reproducibility, and epidemiological concordance were determined for isolates belonging to both serogroup 1 and the other serogroups investigated. Gene fragments were amplified from genomic DNA, and PCR amplicons were sequenced by using forward and reverse primers. Consensus sequences are entered into an online database, which allows the assignment of individual allele numbers. The resulting sequence-based type or allelic profile comprises a string of the individual allele numbers separated by commas, e.g., 1,4,3,1,1,1, in a predetermined order, i.e., flaA, pilE, asd, mip, mompS, and proA. The index of discrimination (D) obtained with these six loci was calculated following analysis of a panel of 79 unrelated clinical isolates. A D value of > 0.94 was obtained, and this value appears to be sufficient for use in the epidemiological investigation of outbreaks caused by L. pneumophila. The D value rose to 0.98 when the results of the analysis were combined with those of monoclonal antibody subgrouping. Sequence-based typing of L. pneumophila is epidemiologically concordant and discriminatory, and the data are easily transportable. This consensus method will assist in the epidemiological investigation of L. pneumophila infections, especially travel-associated cases, by which it will allow a rapid comparison of isolates obtained in more than one country.     

Ultrastructural,histochemical, and freeze-fracture evaluation of multilamellated structures in human pulmonary alveolar proteinosis

Ultrastructural, histochemical, and freeze-fracture studies of material recovered by bron-choalveolar lavage from patients with pulmonary alveolar proteinosis revealed four types (A, B, C, and D) of multilamellated structures (MS). Type A, the major component, consisted of concentric, trilaminar structures which were composed of two electron-dense layers and a central lucent layer (5.7–7.5 nm in overall width) alternating with wider (25–30 nm) electron-lucent intervening layers. Type B MS were formed by concentric lamellae with a 5–5.3-nm periodicity. Type C MS were composed of wavy, electron-dense lamellae with a 4–4.5-nm periodicity. Type D MS were conglomerated masses of intricately arranged double or triple electron-dense layers (7.5–13.5 nm wide) alternating with wider (30–40-nm) electron-lucent layers. The electron-dense lamellae of type A, type C, and type D MS were stained with ruthenium red, the Thiéry method, and concanavalin A, indicating the presence of carbohydrate components. Freeze-fracture studies revealed smooth inner and outer surfaces in type A MS, with the fracture planes passing through the central parts of the trilaminar structures; the intervening layers contained 10-nm particles, which probably are proteins. Type B MS had smooth surfaces, and type C MS had slightly particulate surfaces; while type D MS showed tubular or polygonal structures, 350 nm wide, with rows of particles 7–8 nm in diameter. It is concluded that type A and type D MS contain proteins and carbohydrates, probably in the form of glycoproteins, as well as phos-pholipids, and are related to tubular myelin. Type B and type C MS are considered to contain mainly phos-pholipids; type C MS are also considered to contain carbohydrates and to be related to lamellar bodies of type II alveolar epithelial cells.     

Histochemical and immunohistochemical study of diffuse large-cell lymphomas.

Ninety diffuse large-cell lymphomas (diffuse histiocytic lymphoma) were subclassified into B-cell, T-cell, and histiocytic types according to their enzyme histochemical and immunohistochemical characteristics. The B-cell type was characterized by presence of intracellular monoclonal immunoglobulin; negative or weakly positive diffuse acid phosphatase activity; and an occasional focal nodular pattern or preceding nodular lymphoma. The T-cell type was characterized by moderate, focal acid phosphatase activity; convoluted nuclear structure; and frequent preceding cutaneous manifestations. The histiocytic type was characterized by strong nonspecific esterases and diffuse acid phosphatase activity and presence of lysozyme and phagocytic activity. Most of the lesions (74 cases) were of the B-cell type. This group was further subdivided into follicular center cell type and B-cell immunoblastic sarcoma, according to the stage of cellular transformation. Preliminary clinical correlation suggests that the histiocytic type is most resistant to treatment. B-cell immunoblastic sarcomas were much more aggressive than neoplasms of the follicular center cell type.     

Role of volume-stimulated osmolyte and anion channels in volume regulation by mammalian sperm

The ability to maintain cellular volume is an important general physiological function. Swelling induced by hypotonic stress results in the opening of channels, through which ions exit with accompanying water loss (regulatory volume decrease, RVD). RVD has been shown to occur in mammalian sperm, primarily through the opening of quinine-sensitive potassium channels. However, as yet, direct evidence for the participation of anion channels in sperm RVD has been lacking. The chloride channel type ClC-3 is believed to be involved in RVD in other cell types. Using electronic cell sizing for cell volume measurement, the following results were obtained. (i) The anion channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), tamoxifen and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) increased hypotonic swelling in concentration-dependent fashion, whereas verapamil (P-glycoprotein inhibitor) had little effect. The most potent, NPPB and DIDS, blocked RVD without affecting cell membrane integrity at effective concentrations. (ii) When gramicidin was included to dissipate Na+/K+ gradients, major secondary swelling was observed under hypotonic conditions. This secondary swelling could be reduced by NPPB, and suppressed completely by replacing chloride in the medium with sulphate, an ion which does not pass through chloride channels. It was deduced that the initial hypotonic swelling activated an anion channel through which chloride ions could then enter freely down a concentration gradient, owing to the lack of a counter-gradient of potassium. (iii) Taurine, an osmolyte often involved in RVD, does not appear to play a role in sperm RVD because lengthy preincubation with taurine did not alter sperm RVD response. Our observations provide direct evidence that a chloride channel (possibly ClC-3) is involved in the process of volume regulation in mammalian sperm.     

How useful is weight reduction in the management of hypertension?

A group of previously untreated obese hypertensive patients were started on a weight reduction programme supervised by two dietitians working in a general practice surgery. It was stressed from the beginning of the programme that reducing blood pressure was the purpose of the diet. The results of follow-up after six months are presented together with results for a control group of obese hypertensive patients not receiving dietary advice or drug therapy, but being followed by the general practitioner. The weight, systolic blood pressure and diastolic blood pressure of the dieting hypertensive group were significantly lower than those of the non-dieting group after six months. However, the drop-out rate was significantly higher for the dieting group than for the non-dieting group.

The results of a separate comparison between a control group of obese normotensive patients following the same dietary programme and the group of dieting obese hypertensive patients are also presented. Attendance rates and weight loss achieved were significantly better for the hypertensive group than for the normotensive group after 12 months.

Weight reduction appears to be an effective first-line therapy for approximately 50% of obese patients with mild to moderate hypertension, and raised blood pressure appears to provide motivation for such patients to attend a dietitian's clinic and to lose weight.

     

Quality assurance programme for necropsies.

One hundred and eight consecutive necropsies were entered into a quality assessment of the necropsy service in this hospital using a previously published American protocol. Our results were similar to those of the American series, with confirmation of the major clinical diagnosis in 75%, but a higher rate of unsuspected diagnosis (44%). Necropsy was helpful in 58% of cases. The presentation of selected cases at a monthly conference on causes of death was valuable in correlating clinical and pathological findings, and in helping integrate both services.     

Colonization and maintenance of murine embryonic stem cells on poly(alpha-hydroxy esters)

The aim of this study was to determine the ability of various poly(alpha-hydroxy esters) to support the in vitro propagation of murine embryonic stem (ES) cells in an undifferentiated state. To this end, ES cell colonization, growth and Oct-4 immunoreactivity following a 48 h culture period upon poly((D,L)-lactide), poly((L)-lactide), poly(glycolide) and poly((D,L)-lactide-co-glycolide) (PLGA) were assessed. By the analysis of live and dead cell number indices and Oct-4 immunoreactivity, ES cell colonization rate during a 48 h culture period was found to be significantly greater on PLGA compared to all the other unmodified poly(alpha-hydroxy esters) tested. Surface treatment of all polymers with 0.1m potassium hydroxide revealed a significant increase in ES cell live numbers when compared to all unmodified polymers, thus revealing a correlation between polymer content, hydrophilicity and colonization rate. These data suggest that surface treated poly(alpha-hydroxy esters) may be employed for ES cell scale up procedures and in tissue engineering applications requiring the colonization of scaffolds by ES cells in an undifferentiated state. According to such applications, once the designated scaffold has been colonized, ES cell directed differentiation into the desired and fully differentiated, functional adult tissue may then be effected.     

Differential diagnosis of Taenia saginata and Taenia solium infection by PCR

We have designed species-specific oligonucleotides which permit the differential detection of two species of cestodes, Taenia saginata and Taenia solium. The oligonucleotides contain sequences established for two previously reported, noncoding DNA fragments cloned from a genomic library of T. saginata. The first, which is T. saginata specific (fragment HDP1), is a repetitive sequence with a 53-bp monomeric unit repeated 24 times in direct tandem along the 1, 272-bp fragment. From this sequence the two oligonucleotides that were selected (oligonucleotides PTs4F1 and PTs4R1) specifically amplified genomic DNA (gDNA) from T. saginata but not T. solium or other related cestodes and had a sensitivity down to 10 pg of T. saginata gDNA. The second DNA fragment (fragment HDP2; 3,954 bp) hybridized to both T. saginata and T. solium DNAs and was not a repetitive sequence. Three oligonucleotides (oligonucleotides PTs7S35F1, PTs7S35F2, and PTs7S35R1) designed from the sequence of HDP2 allowed the differential amplification of gDNAs from T. saginata, T. solium, and Echinococcus granulosus in a multiplex PCR, which exhibits a sensitivity of 10 pg.