Detection of Bacteroides fragilis Enterotoxin Gene by PCR

Bacteroides fragilis constitutes about 1% of the bacterial flora in intestines of normal humans. Enterotoxigenic strains of B. fragilis have been associated with diarrheal diseases in humans and animals. The enterotoxin produced by these isolates induces fluid changes in ligated intestinal loops and an in vitro cytotoxic response in HT-29 cells. We developed a nested PCR to detect the enterotoxin gene of B. fragilis in stool specimens. After DNA extraction, a 367-bp fragment was amplified with two outer primers. The amplicon from this reaction was subjected to a second round of amplification with a set of internal primers. With these inner primers, a 290-bp DNA fragment was obtained which was confirmed as part of the B. fragilis enterotoxin gene by Southern blotting with a nonradioactive internal probe and a chemiluminescence system. By this approach, B. fragilis enterotoxin gene sequences were detected in eight known enterotoxigenic human isolates and nine enterotoxigenic horse isolates. No amplification products were obtained from DNA extracted from 28 nonenterotoxigenic B. fragilis isolates or B. distasonis, B. thetaiotaomicron, B. uniformis, B. ovatus, Escherichia coli, or Clostridium difficile. The sensitivity of this assay allowed us to detect as little as 1 pg of enterotoxin DNA sequences or 100 to 1,000 cells of enterotoxigenic B. fragilis/g of stool. Enterotoxin production of all isolates was confirmed in vitro in HT-29 cells. A 100% correlation was obtained between enterotoxin detection by cytotoxin assay and the nested PCR assay. This rapid and sensitive assay can be used to identify enterotoxigenic B. fragilis and may be used clinically to determine the role of B. fragilis in diarrheal diseases.     

Unusual retinal manifestation in a combination of Sturge-Weber and Klipplel-Trenaunay syndrome--a case report

An unusual retinal manifestation of arteriovenous communications is reported in a case showing a combination of Sturge-Weber and Kippel-Trenaunay syndromes. The arteriovenous communications are thought to be similar to those seen in the limbs in Parkes-Weber syndrome. This retinal finding indicates that the three syndromes are intricately related to each other and may be an incomplete manifestation of a single pathophysiologic entity which may be named neurocutaneous angiomatosis.     

A call for a gender specific approach to address the worldwide cardiovascular burden

Cardiovascular diseases (CVD) among non-communicable diseases are already a major public health challenge worldwide. A further increase in CVD is projected to occur over the next 25 years as a result of both adverse lifestyle changes and demographic shifts in the population age profile. The adverse impact of these health problems will affect women in particular, given the steady rise in the proportion of the aging population that will be women.The critical issue presently in the management of CVD is that we are not even adequately using the data that are available. Women still remain unaware that they are at risk, and information about women is not easily accessible to their physicians. This is a global issue and the need remains for worldwide initiatives with greater vigilance to identify these factors and make efforts to control them effectively.Currently, in scientific research, it is expected that the results of clinical research be analyzed for sex differences, sex- and gender-appropriateness, and sex- and gender-specific approaches for prevention, diagnosis, treatment, and counseling. To address the care discrepancy, the global community needs to develop a conducive environment within a comprehensive policy and operational framework to achieve favorable lifestyles, and CVD risk factor reduction for both men and women.     

Typhus and typhuslike rickettsiae associated with opossums and their fleas in Los Angeles County, California.

The recent discovery of cat fleas (Ctenocephalides felis) infected with a typhuslike rickettsia (designated the ELB agent) raises the question of whether similar rickettsial infections exist in wild cat flea populations. We verified the presence of the ELB agent and Rickettsia typhi in urban and suburban areas of Los Angeles, Calif. Opossums trapped in close proximity to the residences of human murine typhus cases in Los Angeles county and other areas within the city of Los Angeles were tested for the presence of typhus group rickettsiae by the polymerase chain reaction (PCR). The presence of rickettsiae in the spleen tissues of three opossums (n = 9) and in 66 opossum fleas (n = 205) was determined by PCR and was verified by dot blot and Southern transfer hybridization. Further analysis of the amplified PCR products generated by a series of primer pairs derived from either the 17-kDa antigen gene or the citrate synthase gene revealed that both R. typhi and the ELB agent were present in the tested samples. Dual infection was not noted in the samples; however, the fleas were infected with either R. typhi or the ELB agent. The presence of the ELB agent in the cat flea population may have implications for public health. Whether this agent is responsible for the mild cases of human murine typhus in urban and suburban areas of Los Angeles or in other endemic foci remains to be determined.     

Rickettsia-macrophage interactions: host cell responses to Rickettsia akari and Rickettsia typhi

The existence of intracellular rickettsiae requires entry, survival, and replication in the eukaryotic host cells and exit to initiate new infection. While endothelial cells are the preferred target cells for most pathogenic rickettsiae, infection of monocytes/macrophages may also contribute to the establishment of rickettsial infection and resulting pathogenesis. We initiated studies to characterize macrophage-Rickettsia akari and -Rickettsia typhi interactions and to determine how rickettsiae survive within phagocytic cells. Flow cytometry, microscopic analysis, and LDH release demonstrated that R. akari and R. typhi caused negligible cytotoxicity in mouse peritoneal macrophages as well as in macrophage-like cell line, P388D1. Host cells responded to rickettsial infection with increased secretion of proinflammatory cytokines such as interleukin-1beta (IL-1beta) and IL-6. Furthermore, macrophage infection with R. akari and R. typhi resulted in differential synthesis and expression of IL-beta and IL-6, which may correlate with the existence of biological differences among these two closely related bacteria. In contrast, levels of gamma interferon (IFN-gamma), IL-10, and IL-12 in supernatants of infected P388D1 cells and mouse peritoneal macrophages did not change significantly during the course of infection and remained below the enzyme-linked immunosorbent assay cytokine detection limits. In addition, differential expression of cytokines was observed between R. akari- and R. typhi-infected macrophages, which may correlate with the biological differences among these closely related bacteria.     

Rat leucocyte response to the bites of rat fleas (Siphonaptera: Pulicidae)

The host response to bites of the oriental rat flea, Xenopyslla cheopis Rothschild, was investigated by examining rat blood leucocyte kinetics, histopathology, and the effect that the host response had upon subsequent flea feeding and longevity. Test rats were subjected to controlled exposures of fleas, and leucocyte data from test rats were compared to those of unexposed controls. Of the five leucocyte types examined, only the basophil appeared to play a role in the host blood response to flea bites. Significant increases in blood basophil levels occurred 2-3 d after exposure but subsided to control levels within a week. However, flea feeding did not produce histopathology at the flea feeding sites nor did the basophilic blood response of rats affect subsequent feeding or longevity of the fleas.     

Vλ-light chain genes reconstitute immune responses to defined carbohydrate antigens or haptens by utilizing different VH genes

The contribution of the λ-light chain to the development of peripheral B cell repertoire and generation of specific antibodies to haptens and polysaccharide antigens was studied in genetically manipulated kappa-deficient and λ₂-transgenic mice. The results clearly demonstrate a non-stoichiometric V_H gene family expression in the absence of k-light chain and suggest a non-stochastic pairing between V_H and V_λ genes, expressed in the peripheral B cell repertoire. A shift in V_H gene utilization in the case of Vl_λ⁺ antibodies was evident in response to β2–6 fructosan and TNP hapten. These observations demonstrate the availability of compensatory mechanisms in the absence of V_K genes and are consistent with the hypothesis that V_H gene family expression is controlled by genetic factors from inside the V_H locus. Furthermore, genetic factors from outside the V_H locus, namely restricted available light chain diversity, may lead to a shift in V_H gene utilization in the peripheral B cell repertoire.