Role of T cell costimulation in anti-viral immunity

Members of both the CD28 and TNFR families can have costimulatory roles in T cell activation. Gene targeted mice as well as in vivo blocking experiments have established distinct roles for CD28/B7; ICOS/ICOSL; CD27/CD70; 4-1BB/4-1BBL and OX40/OX40L during viral infection. Many issues remain to be addressed, including the timing and location of the interactions, the possibility of partial redundancy between related family members and the molecular basis for the specific phenotypes observed in the different gene targeted mice.     

Rapid DNA haplotyping using a multiplex heteroduplex approach: Application to duchenne muscular dystrophy carrier testing

A new strategy has been developed for rapid haplotype analysis based on an initial multiplex amplification of several polymorphic sites, followed by heteroduplex detection. Heteroduplexes formed between two different alleles are detected because they migrate differently than the corresponding homoduplexes in Hydrolink-MDE gel. This simple, rapid method does not depend on specific sequences such as restriction enzyme sites or CA boxes and does not require the use of isotope. This approach has been tested using commonly occurring polymorphisms spanning the dystrophin gene as a model. We describe the use of the method to assign the carrier status of females in Duchenne muscular dystrophy (DMD) pedigrees. The method may be used for other genetic diseases when mutations are unknown or there are few dinucleotide markers in the gene proximity, and for the identification of haplotype backgrounds of mutant alleles. © 1995 Wiley-Liss, Inc.     

Chimerism studies in HLA-identical nonmyeloablative hematopoietic stem cell transplantation point to the donor CD8(+) T-cell count on day + 14 as a predictor of acute graft-versus-host disease.

Chimerism analysis of hematopoietic cells has emerged as an essential tool in nonmyeloablative hematopoietic stem cell transplantation. We have investigated the development of donor chimerism in granulocytes and CD4(+) and CD8(+) T cells in blood and bone marrow of 24 patients with hematologic malignancies who received HLA-identical sibling peripheral blood stem cell grafts after conditioning with fludarabine and 2 Gy of total body irradiation. The T-cell chimerism of blood and bone marrow was tightly correlated. Complete donor chimerism was reached earlier in the granulocytes than in the T cells. Mixed T-cell chimerism was common at the time of onset of acute graft-versus-host disease (aGVHD), and both CD4(+) and CD8(+) donor T-cell chimerism increased with the occurrence of aGVHD grades II to IV (P =.0002 and P =.019, respectively). The rate of disappearance of recipient CD8(+) T cells was faster in patients with aGVHD grades II to IV than in patients without clinically significant aGVHD (P =.016). This observation indicates a role of graft-versus-lymphohematopoietic tissue reactions in creating complete donor T-cell chimerism. A donor CD8(+) T-cell count above the median on day +14 increased the risk of subsequent development of aGVHD grades II to IV (P =.003).     

Role of depth in eye distance measurements: Comparison of single and stereo‐photogrammetry

Photogrammetry has been used as an alternative to direct measurements to obtain facial distances for a variety of anthropometric applications. Taking measurements from photographs is less intrusive to subjects and reduces screening time, but measurements from single frontal photographs neglect depth information and may be inadequate for screening purposes. This study examined the role of depth in measurements of palpebral fissure length, interpupillary distance, inner canthal distance, and outer canthal distance using single‐ and stereo‐photogrammetry; an operator selected landmarks on single and stereo digital photographs displayed on a computer monitor. Depth was not found to make a significant contribution to eye distances in an idealized system where the real‐world coordinates of points on the eye were known from three‐dimensional calibration of stereo photographs. However, the differences found between measurements taken from single frontal photographs and those from stereo‐photogrammetry indicated that measurements from single photographs are prone to errors due to misalignment of the camera, the face, and the calibration instrument during image acquisition; if single photographs are to be used, the placement of these components should be carefully monitored. Am. J. Hum. Biol. 15:573–578, 2003. © 2003 Wiley‐Liss, Inc.     

CD1d-restricted NKT cells contribute to malarial splenomegaly and enhance parasite-specific antibody responses

CD1d-restricted NKT cells are a novel T cell lineage with unusual features. They co-express some NK cell receptors and recognize glycolipid antigens through an invariant T cell receptor (TCR) in the context of CD1d molecules. Upon activation through the TCR, NKT cells produce large amounts of IFN-gamma and IL-4. It has been proposed that rapid cytokine output by activated NKT cells may induce bystander activation of other lymphoid lineages. The impact of CD1d-restricted NKT cell activation in the induction of B cell-mediated immune responses to infection is still unclear. We show here that CD1-restricted NKT cells contribute to malarial splenomegaly associated with expansion of the splenic B cell pool and enhance parasite-specific antibody formation in response to Plasmodium berghei infection. The increased B cell-mediated response correlates with the ability of NKT cells to promote Th2 immune responses. Additionally, antibody responses against the glycosylphosphatidylinositol (GPI)-anchored protein merozoite surface protein 1 (MSP-1) were found to be significantly lower in CD1(-/-) mice compared to wild-type animals. P. berghei-infected MHC class II (MHCII)(-/-) mice also generated antibodies against MSP-1, suggesting that antibody production against GPI-anchored antigens in response to malaria infection can arise from both MHCII-dependent and independent pathways.     

Expression and function of 4-1BB during CD4 versus CD8 T cell responses in vivo

4-1BBL(-/-) mice have a defect in recall CD8+ T cell responses to viruses, whereas CD4+ T cell responses to virus are unimpaired in these mice. In contrast, both CD4+ and CD8+ T cells respond to 4-1BB ligand (4-1BBL) in vitro. To clarify the role of 4-1BB/4-1BBL in CD4+ versus CD8+ T cell responses in vivo, we compared CD4 (OT-II) and CD8 (OT-I) TCR transgenic T cells responding to the same antigen in an in vivo adoptive transfer model in 4-1BBL(+/+) versus 4-1BBL(-/-) mice. During primary and secondary responses, expression of 4-1BB on in vivo-activated TCR transgenic T cells was earlier and more transient than previously observed in vitro, correlating with expression of the early activation antigen CD69 and preceding the transition to the CD44hi state. Although 4-1BB is expressed early in the primary response, there was no effect of 4-1BBL deficiency on initial CD8 T cell expansion and only a minor effect on initial CD4 T cell expansion. The major effect of 4-1BB/4-1BBL interaction is on the T cell recall response. This is due to effects of 4-1BBL on maintenance of T cell numbers at the end of the primary response with additional effects of 4-1BBL on secondary expansion of T cells.     

Heterogeneity in the plasma levels of two acute-phase proteins in mice from inbred strains infected withTrypanosoma cruzi

We analyzed the variations observed in the plasma levels of both alpha-macroglobulins (AM) and serum amyloid P (SAP) in mice from three different inbred strains (C3H, Balb/C and C57black/6) acutely infected withTrypanosoma cruzi. SAP levels increased in C57black/6 and Balb/C mice but not C3H mice. AM levels increased in all C3H mice but not in C57black/6 mice and rose slightly in only 43% of the Balb/C mice. AM and SAP levels are differently modulated in patterns that may be strain-determined.     

Adherence Patterns and Adherence-Related DNA Sequences in Escherichia coli Isolates from Children with and without Diarrhea in S?o Paulo City,Brazil

The correlation between various adherence patterns and adherence-related DNA sequences in Escherichia coli isolates from 1- to 4-year-old children with and without diarrhea in São Paulo, Brazil, was evaluated. A total of 1,801 isolates obtained from 200 patients and 200 age-matched controls were studied. The adherence patterns found were classified as diffuse, aggregative, aggregative in a 6-h assay, aggregative predominantly in coverslips, localized, localized-like, and noncharacteristic. In general, the DNA sequences used as probes showed excellent specificities (>93%), but their sensitivities varied. Thus, the results of bioassays and assays with DNA probes normally used to search for adherent E. coli did not correlate well, and the best method for the identification of these organisms in the clinical research setting remains controversial. Isolates presenting diffuse adherence or hybridizing with the related daaC probe, or both, were by far the most frequent in patients (31.5, 26.0, and 23.0%, respectively), followed by isolates presenting aggregative adherence or hybridizing with the related EAEC probe, or both (21.5, 13.0, and 10.5%, respectively). None of the different combinations of adherence patterns and adherence-related DNA sequences found were associated with acute diarrhea.The first step in the establishment of the diarrheal diseases caused by the various categories of diarrheagenic Escherichia coli is adherence to epithelial cells of the intestinal mucosa. In vitro assays with eukaryotic cell lines (HeLa and HEp-2 cells) have identified three distinct adherence patterns among fecal isolates of E. coli: localized, diffuse, and aggregative (37, 38, 41). Localized adherence (LA) is characterized by formation of bacterial microcolonies on a restricted area(s) of the cell surface, while diffuse adherence (DA) is the scattered attachment of bacteria over the whole surface of the cell (41). The pattern of aggregative adherence (AA) consists of bacterial attachment to the cells and the intervening cell growth surface in a stacked brick-like lattice (37).The LA pattern was first detected in strains classified as enteropathogenic E. coli (EPEC) among serogroups associated with outbreaks of infantile diarrhea (41). Although E. coli strains exhibiting DA (DAEC) have been isolated at similar frequencies from feces of infants and young children with acute diarrhea and nondiarrheic controls in some populations (3, 10, 11, 14, 18), they were significantly associated with diarrhea in other settings (1, 17, 24, 29, 33). E. coli strains showing AA, termed enteroaggregative E. coli (EAEC), have been linked to sporadic persistent diarrhea (3, 4, 7, 10, 13, 26, 27, 44) and to outbreaks of diarrhea in both developing and developed countries (8, 12, 28, 43). However, the role of EAEC in acute diarrhea is still controversial: some studies have shown a correlation (7, 23, 25, 27, 34, 37), but others (1, 3, 6, 10, 11, 13–15, 17, 18, 24, 26, 29, 33, 44) have not.DNA probes derived from adherence-related sequences have been constructed (2, 5, 16, 31, 36) and used in hybridization assays for the detection of the different established and putative categories of diarrheagenic E. coli in many epidemiological studies.We evaluated the relationship between the LA, DA, and AA patterns and hybridization with adherence-related DNA sequences and tested children 1 to 4 years old with and without acute diarrhea for the presence of adherent E. coli strains.     

Effects of Bovine Colostrum with or without Egg on In Vitro Bacterial-Induced Intestinal Damage with Relevance for SIBO and Infectious Diarrhea

Small intestinal bacterial overgrowth (SIBO) occurs commonly, is difficult to treat, and frequently recurs. Bovine colostrum (BC) and chicken eggs contain immunoglobulins and other components that possess antimicrobial, immunoregulatory, and growth factor activities; however, it is not known if they have the ability to reduce injury caused by the presence of bacteria associated with SIBO (Streptococcus, Escherichia coli, Staphylococcus, Bacteroides, Klebsiella, Enterococcus, and Proteus) and infectious diarrhea (enteropathogenic Escherichia coli, Salmonella). We examined the effects of BC, egg, or the combination, on bacterial growth and bacteria-induced changes in transepithelial electrical resistance (TEER) and bacterial translocation across confluent Caco-2 monolayers. BC, egg, or the combination did not affect bacterial growth. Adding bacteria to monolayers reduced TEER and (with minor variations among species) increased bacterial translocation, increased monolayer apoptosis (increased caspase-3 and Baxα, reduced Bcl2), increased intercellular adhesion molecule 1 (ICAM-1), and reduced cell adhesion molecules zonulin1 (ZO1) and claudin-1. BC, egg, or the combination reduced these effects (all p < 0.01) and caused additional increases in vascular endothelial growth factor (VEGF) and Heat Shock Protein 70 (Hsp70) expression. We conclude that BC ± egg strengthens mucosal integrity against a battery of bacteria relevant for SIBO and for infectious diarrhea. Oral BC ± egg may have clinical value for these conditions, especially SIBO where eradication of precipitating organisms may be difficult to achieve.