Group C rotavirus associated with fatal enteritis in a family outbreak

A family outbreak of gastroenteritis involving three adults and three children is described in which diarrhoea and vomiting were the main clinical features. One infant died in whom no pathogens could be detected in either small or large intestinal postmortem samples. Stool samples from two symptomatic siblings contained rotaviruses as demonstrated by electron microscopy. Both of these faecal samples were negative when assayed in a group A specific rotavirus enzyme-linked immunosorbent assay (ELISA) and subsequent genomic analysis of these rotaviruses was suggestive of group C rotavirus. Serological evidence showed that these atypical rotaviruses were members of serogroup C. Other atypical rotaviruses in faecal samples from sporadic cases in symptomatic children were detected over a similar time period and location. These had electrophoretic RNA profiles similar to those in the family outbreak. Furthermore, seroepidemiological studies detected group C rotavirus antibody in blood donors resident in the location of the family outbreak.     

Apoptosis induction and interferon signaling but not IFN-beta promoter induction by an SV5 P/V mutant are rescued by coinfection with wild-type SV5

Infection of human cells with the paramyxovirus simian virus 5 (SV5) results in minimal cytopathic effect, and host interferon (IFN) and apoptotic pathways are not activated. We have previously shown that an rSV5 containing six naturally occurring P/V gene substitutions (rSV5-P/V-CPI-) displays premature and elevated expression of viral RNA and protein. In addition, cells infected with rSV5-P/V-CPI- show induction of the IFN-beta promoter as well as activation of IFN signaling and apoptotic pathways. In this article, we have tested the hypothesis that rSV5-WT can supply trans-acting factors that prevent host cell antiviral responses induced by rSV5-P/V-CPI-. During coinfection of human A549 cells, rSV5-WT blocked cell rounding, loss of cell volume, and DNA fragmentation induced by rSV5-P/V-CPI-, three later events in the apoptotic pathway, but was not able to block the loss of mitochondrial membrane potential (DeltaPsi(m)), an early event in the cell death process. As expected, IFN signaling was blocked during coinfections, and this was attributed to the loss of STAT1 induced by the rSV5-WT V protein. Surprisingly, simultaneous infection with rSV5-WT could not suppress the activation of the IFN-beta promoter by rSV5-P/V-CPI- infection. However, the IFN-beta promoter was not activated in cells that were first preinfected for 1 h with rSV5-WT and then subsequently infected with rSV5-P/V-CPI-. A model is proposed for activation of host responses to infection with the rSV5-P/V-CPI- mutant and the steps that are blocked by rSV5-WT.     

Frequency of cytokine polymorphisms in populations from western Europe,Africa, Asia,the Middle East and South America

PCR-SSOP identification procedures for IL-2, IL-6, IL-10, TNF-alpha and TNF-beta cytokine polymorphisms have been developed. Application of the procedures to a range of diverse geographically distributed populations has identified ethnic differences within the groups studied. Five populations were investigated, Northern Ireland, South African Zulu, Omani, Singapore Chinese and Mexican Mestizos.     

Growth sensitivity of a recombinant simian virus 5 P/V mutant to type I interferon differs between tumor cell lines and normal primary cells

A paramyxovirus SV5 mutant (rSV5-P/V-CPI-) that encodes 6 naturally-occurring P/V gene substitutions is a potent inducer of type I interferon (IFN) and is restricted for low moi growth, two phenotypes not seen with WT SV5. In this study, we have compared the IFN sensitivity of WT SV5 and the rSV5-P/V-CPI- mutant in tumor cell lines and in cultures of normal primary cells. We have tested the hypothesis that differences in IFN induction elicited by WT rSV5 and rSV5-P/V-CPI- are responsible for differences in low moi growth and spread. In contrast to WT SV5, low moi infection of A549 lung carcinoma cells with rSV5-P/V-CPI- resulted in a plateau of virus production by 24-48 h pi when secreted IFN levels were between approximately 100 and 1000 U/ml. Gene microarray and RT-PCR analyses identified IFN genes and IFN-stimulated genes whose expression were increased by infection of A549 cells with WT and P/V mutant viruses. Restricted low moi growth and spread of rSV5-P/V-CPI- in A549 cells was relieved in the presence of neutralizing antibodies to IFN-beta but not TNF-alpha. When A549 or MDA-MB-435 breast tumor cells were pretreated with IFN, both WT and P/V mutant viruses showed delayed spread and approximately 10-fold reduction in virus yield, but infections were not eliminated. Using normal primary human epithelial cells that have undergone limited passage in culture, WT rSV5 and rSV5-P/V-CPI- displayed high moi growth properties that were similar to that seen in A549 cells. However, IFN pretreatment of these primary cells as well as normal human lung cells eliminated low moi spread of both mutant and WT rSV5 infections. Together, these data demonstrate that SV5 growth in normal primary human cells is highly sensitive to IFN compared to growth in some tumor cell lines, regardless of whether the P/V gene is WT or mutant. These results suggest a model in which spread of WT SV5 in normal human cells is dependent on the ability of the virus to prevent IFN synthesis. The implications of these results for the use of recombinant paramyxoviruses as vectors are discussed.     

Selective elimination of alloreactive donor T cells attenuates graft-versus-host disease and enhances T-cell reconstitution.

Impaired T-cell immune reconstitution is a major complication after allogeneic bone marrow transplantation (BMT) and is particularly exacerbated in the setting of graft-versus-host disease (GVHD). Conventional approaches to reduce GVHD, such as T-cell depletion or pharmacologic immunosuppression, typically fail to enhance T-cell immunity and often further exacerbate this problem. An alternative strategy to mitigate GVHD severity is the selective elimination of graft-versus-host-reactive donor T cells by using an incorporated thymidine kinase suicide gene. This approach has been shown to effectively reduce GVHD, although the effect of this strategy on T-cell reconstitution is unresolved. We addressed this question in a murine BMT model (C57BL/6 [H-2(b)] --> AKR/J [H-2(k)]) in which donor and recipient differ at major and minor histocompatibility antigens. Lethally irradiated AKR recipients transplanted with T cell-depleted bone marrow plus thymidine kinase-positive T cells followed by post-BMT ganciclovir (GCV) administration had more prompt and complete normalization of the T-cell repertoire than phosphate-buffered saline-treated GVHD control animals. By 60 days after transplantation, mice administered GCV had T-cell repertoires that were virtually indistinguishable from those of mice that underwent transplantation with T cell-depleted bone marrow alone (no GVHD controls) when assayed by T-cell receptor (TCR) spectratyping. In contrast, phosphate-buffered saline-treated animals had persistent skewing in most Vbeta families. T cells obtained from GCV-treated mice also had significantly higher in vitro proliferative responses after posttransplantation inoculation with ovalbumin than GVHD animals, indicating that CD4(+) T-cell responses against a nominal antigen were better preserved in these chimeras. Finally, GCV-treated mice had augmented immune reconstitution in response to exogenous interleukin-7 administration, as evidenced by increased overall spleen cellularity and absolute numbers of T and B cells. This was in contrast to GVHD control animals, which had a blunted response to interleukin-7 administration. These data indicate that GVHD severity can be significantly reduced by selective elimination of alloreactive donor T cells without compromise of T-cell immunity. Moreover, in light of previous studies demonstrating that this strategy can reduce GVHD without loss of alloengraftment and antileukemia reactivity, further examination of this approach in humans seems warranted.     

The use of a standard proforma in breast cancer reporting

AIM: To determine whether the introduction of a standard reporting proforma has led to an improvement in the completeness of histopathology reports for breast cancer excision specimens. METHODS: A standard reporting proforma was designed using the Royal College of Pathologists' minimum dataset for breast cancer histopathology reports and the national histopathology reporting form of the National Health Service (NHS) breast screening programme. This was introduced into our department in June 1999, with reports generated from the proforma replacing the standard text reports. The pathological information contained in 50 text reports issued before the introduction of the proforma and 50 reports generated using the proforma was compared with the minimum dataset and NHS breast screening programme guidelines. RESULTS: A general improvement in documentation of individual pathological features was noted after introduction of the proforma. This was most significant in relation to documentation of features, such as microcalcification and ductal carcinoma in situ. In addition, important features such as tumour grade, tumour size, and hormone receptor status were documented more frequently in the proforma group. There was an overall increase in the number of reports regarded as complete after introduction of the proforma. CONCLUSIONS: The introduction of a standard proforma led to a significant improvement in the completeness of breast cancer histopathology reports in this centre, but continued vigilance is needed to ensure that standards continue to improve.     

Molecules relevant for T cell-target cell interaction are present in cytolytic granules of human T lymphocytes

An ultrastructural analysis of human cytotoxic T lymphocyte-target cell (CTL-TC) interaction has been undertaken to enable a better understanding of the killing mechanism. Attention was focused on granules in the CTL, which are known to contain lethal compounds. Within the membrane-delimited cytotoxic granule an electron-dense core as well as numerous membrane vesicles were identified. In CTL-TC conjugates, specific membrane interactions take place, allowing the formation of intercellular clefts into which the granule cores and internal vesicles are released. T cell surface membrane molecules known to be involved in CTL-TC interaction (T cell receptor, CD3 and CD8) are present on the membranes of the granule cores and internal vesicles, facing outward. An explanation for this localization of the membrane may be found in the fact that the granule is connected with an endocytotic pathway. Moreover, the lumen of the granule is rich in the enzyme cathepsin D, which indicates an association with a lysosomal compartment. Exocytosed vesicles and cores are seen to adhere to the plasma membrane of the TC. Although the exact contents of the granule vesicles and core remain to be identified, we suggest that specific interaction of CTL membrane molecules on the cytolytic granule components with molecules on the plasma membrane of the TC may ensure the unidirectional delivery of the lethal hit.     

alpha(v)beta(3) Integrin in central nervous system tumors

alpha(v)beta(3) Is an integrin specifically expressed in endothelial cells of newly forming blood vessels. Integrin-mediated angiogenesis is hypothesized to play a central role in the development and the progression of central nervous system neoplasms. Accordingly, it is considered a potential target for antiangiogenic therapy. In the current study, we compare the expression of alpha(v)beta(3) in ependymomas, oligodendrogliomas, pilocytic astrocytomas, medulloblastomas, and vestibular schwannomas (acoustic neuromas). Samples of 5 tumors of each of the 5 tumor types were harvested surgically and frozen. After the pathological diagnosis was confirmed, immunohistochemistry was performed using an anti- alpha(v)beta(3) monoclonal antibody (LM609). The expression of alpha(v)beta(3) was assessed using a 4-tiered (0-3) grading scheme reflecting the percentage of positively staining vessels. All vestibular schwannomas demonstrated strong (grade 3) alpha(v)beta(3) expression. The expression was uniformly prominent in Antoni B regions of the tumors. Of 5 ependymomas, 4 demonstrated uniformly strong alpha(v)beta(3). Oligodendrogliomas, medulloblastomas, and pilocytic astrocytomas demonstrated more variable alpha(v)beta(3). alpha(v)beta(3) may contribute significantly to angiogenesis in vestibular schwannomas and ependymomas. Despite the high vascular density of oligodendrogliomas, pilocytic astrocytomas, and medulloblastomas, these tumors had variable moderate alpha(v)beta(3) expression. This discrepancy suggests temporal and/or regional variability in the angiogenesis in these types of tumor. This study provides the first demonstration of alpha(v)beta(3) expression in vestibular schwannomas, medulloblastomas, and pilocytic astrocytomas.