Falsely incompatible B-cell flow cytometry crossmatch after pronase treatment: a case report.

This report presents a falsely incompatible B cell crossmatch by flow cytometry in a lung transplant recipient. The patient was a 35-year-old Caucasian male with end-stage lung disease secondary to cystic fibrosis whose pretransplantation serologic workup did not disclose the presence of anti-HLA class II antibodies by single antigen bead testing. Unexpectedly, crossmatch of recipient sera with pronase-treated donor lymphocytes resulted in antibody binding to B cells only. The positive reactivity was reproducible in pronase-treated autologous B cells. Recipient sera did not react with nontreated donor or autologous lymphocytes. Herein, we describe our approach to this unexpected crossmatch result and consider the implications of false-positive crossmatch results on transplantation.

Nitric oxide participates in the intestinal pathology associated with murine syngeneic graft-versus-host disease.

Syngeneic graft-versus-host disease (SGVHD) develops following lethal irradiation, reconstitution with syngeneic bone marrow, and treatment with a short course of cyclosporin A (CsA) therapy. The disease is characterized by the development of a T helper cell type 1-like cytokine response [interleukin (IL)-12, interferon-gamma (IFN-gamma), and tumor necrosis factor alpha], and macrophage activation is central to development of the syndrome. It has been shown that nitric oxide (NO) participates significantly in the development of allogeneic GVHD. Studies were initiated to determine if NO participates in the pathology associated with SGVHD. Significant increases in inducible NO synthase (iNOS) mRNA and circulating NO were found in the tissues of SGVHD versus control animals. Treatment of SGVHD animals with the iNOS inhibitor aminoguanidine (AG) reversed the pathology associated with this disease. Furthermore, AG treatment reduced the production of IL-12 and IFN-gamma mRNA in the colons of CsA-treated mice. These studies demonstrate that NO participates in the pathological processes that are associated with the development of murine SGVHD.

Enhancement or modulation of the vector competence of Ochlerotatus vigilax (Diptera: Culicidae) for ross river virus by temperature.

Two different doses of Ross River virus (RR) were fed to Ochlerotatus vigilax (Skuse), the primary coastal vector in Australia; and blood engorged females were held at different temperatures up to 35 d. After ingesting 10(4.3) CCID50/mosquito, mosquitoes reared at 18 and 25 degrees C (and held at the same temperature) had higher body remnant and head and salivary gland titers than those held at 32 degrees C. although infection rates were comparable. At 18, 25, and 32 degrees C, respectively, virus was first detected in the salivary glands on days 3, 2, and 3. Based on a previously demonstrated 98.7% concordance between salivary gland infection and transmission, the extrinsic incubation periods were estimated as 5, 4, and 3 d, respectively, for these three temperatures. When Oc. vigilax reared at 18, 25, or 32 degrees C were fed a lower dosage of 10(3.3) CCID50 RR/mosquito, and assayed after 7 d extrinsic incubation at these (or combinations of these) temperatures, infection rates and titers were similar. However, by 14 d, infection rates and titers of those reared and held at 18 and 32 degrees C were significantly higher and lower, respectively. However, this process was reversible when the moderate 25 degrees C was involved, and intermediate infection rates and titers resulted. These data indicate that for the strains of RR and Oc. vigilax used, rearing temperature is unimportant to vector competence in the field, and that ambient temperature variations will modulate or enhance detectable infection rates only after 7 d extrinsic incubation. Because of the short duration of extrinsic incubation, however, this will do little to influence RR epidemiology, because by this time some Oc. vigilax could be seeking their third blood meal, the latter two being infectious.

Induction of syngeneic graft-versus-host disease in LPS hyporesponsive C3H/HeJ mice.

Syngeneic GVHD (SGVHD) develops following syngeneic bone marrow transplantation and treatment with cyclosporine A. Previous studies have demonstrated a role for IL-12, IFN-gamma, and TNF-alpha in the development of murine SGVHD. Macrophages can be activated to secrete IL-12 and TNF-alpha via a T-cell-dependent or T-cell-independent pathway (LPS or bacterial products). Studies were designed to determine if LPS participated in the development of SGVHD in C3H/HeN (LPS-responsive) and C3H/HeJ (LPS-hyporesponsive) mice. C3H/HeJ and C3H/HeN mice had similar levels of disease induction and pathology. Following induction of SGVHD, treatment of C3H/HeN, but not C3H/HeJ, mice with a sublethal dose of LPS resulted in mortality. However, neutralization of IL-12 abrogated the development of disease in C3H/HeJ mice, demonstrating that activated macrophages and their products participated in the development of SGVHD in these animals. These data suggested that LPS responsiveness was not a predisposing factor for SGVHD induction.

Report of a new DRB1*13 allele: DRB1*1336.

This brief communication describes the characterization of a new allele, DRB1*1336.

A pilot study of new approaches to teaching anatomy and pathology.

PURPOSE: Minimally Invasive Surgery (MIS) has impacted patient care as well as medical training. New medical education opportunities have emerged with MIS. In this pilot study we explore the role of live, interactive MIS to augment and strengthen specific segments of the undergraduate medical curriculum. METHODS: Laparoscopic cholecystectomy (LC) was selected to demonstrate upper abdominal anatomy and pathology. Second year medical students (n=100) in the course of their GI pathology classes attended live LC telesurgery-the telesurgery student group (TSG). Because of technical difficulties, a second class of medical students (n=90) was shown the tape of the MIS procedure one year later instead of the live surgery-the videotape surgery group (VSG). Background clinical information was provided by the program director and the durgeon. During the live and taped LC broadcast living anatomy was demonstrated and a diseased gallbladder was resected. TSG students were able to ask questions of the program director and the surgeon and vice versa using telesurgery technology. After the procedure, the surgeon met with the students for further discussion. VSG students were able to ask questions of the program director during and after the program. Both groups of students completed a pre- and posttest using remote audience responders. Students' responses from the two groups were compared for selected test and evaluation items. RESULTS: Pre-test (Cronbach's alpha=.10) and post-test (Cronbach's alpha =.28) data were obtained from 73 students in the TSG and.22 and.54 respectively from 69 students in the VSG. A significant increase in laparoscopic anatomy knowledge was observed from pretest to posttest for the VSG (31-55%) and from the TSG (30-61%). The majority of VSG students (68%) indicated the method used to teach was outstanding, and 87% indicated that the program was outstanding in keeping their interest. This is contrasted with only 24% of the TSG group responding that the teaching method was outstanding, and 41% indicated that the program was outstanding in keeping their interest. CONCLUSIONS: Medical students can productively be exposed to surgical methods and living anatomy using telesurgery. The high regard the TSG students had for this program suggests that it can be used effectively to teach and inspire medical students. The positive results have encouraged us to have a backup instructional method such as a tape of the MIS procedure, it apparently does not have the positive impact of live surgery.

Bleomycin-induced pulmonary fibrosis is independent of eosinophils.

Eosinophils have been shown to increase in tissues during many fibrotic conditions and consequently have been suggested to contribute to the development of fibrosis. This study tested the hypothesis that eosinophils are essential in the development of lung fibrosis in mice in response to bleomycin (BLM). Anti-IL-5 antibody was administered intraperitoneally into mice 2 h prior to endotracheal BLM inoculation and thereafter, every other day. Lung eosinophilia was evaluated by measurement of eosinophil peroxidase activity and confirmed by eosinophil counts in histologic sections. Lung fibrosis was evaluated by hydroxyproline content and confirmed by collagen staining in histological sections. Results demonstrated that BLM induced pronounced lung eosinophilia, which was maximal 7 days after BLM treatment and remained elevated through day 14, in C57B1/6 SCID mice and CBA/J mice. In contrast, eosinophilia was a minor component in the lungs of wildtype C57B1/6 mice after BLM treatment, although lung fibrosis developed similarly in all three strains of mice. Treatment with anti-IL-5 completely abrogated eosinophilia but failed to block pulmonary fibrosis induced by BLM in all mouse strains, including C57B1/6 SCID, wildtype C57B1/6 mice, and CBA/J mice. Analysis of cytokine mRNA by RNase-protection assay in C57B1/6 SCID mice indicated that BLM treatment caused enhanced expression of the cytokines, TNF-alpha, and IL-6 at days 3, 7, and 14 post-BLM inoculation, regardless of whether eosinophils were depleted by anti-IL-5. Finally, the importance of eosinophils in lung fibrosis was examined in IL-5 gene knockout mice (IL-5tm1Kopf). BLM treatment induced significant lung fibrosis in IL-5 knockout mice in the absence of eosinophilia. These findings indicate that eosinophils are not an absolute requirement for BLM-induced pulmonary fibrosis in the mouse.

Dissemination barriers to Ross River virus in Aedes vigilax and the effects of larval nutrition on their expression.

Effects of larval nutrition on vector competence of the mosquito Aedes vigilax (Skuse) (Diptera: Culicidae) from Townsville, north Queensland, for Ross River virus (RR) were examined. Larvae were reared on three different diets to create three significantly different size classes of adult mosquito. These were fed on serial dilutions of RR and then sampled on alternate days so the progression of the virus through the mosquito could be examined. No differences of vector competence could be attributed to larval nutrition. Barriers to infection and the correlation between infection rate, viral titre and transmission of RR by Ae. vigilax were also examined. The mesenteronal barrier was the only infection barrier expressed. No correlation between viral titre and transmission was detected, but a strong correlation was found between salivary gland infection and transmission rate. From this it was possible to estimate that 98.7+/-1.3% of Ae. vigilax with infected salivary glands transmit RR.

Th1 cytokines and NK cells participate in the development of murine syngeneic graft-versus-host disease.

Syngeneic graft-vs-host disease (SGVHD) is induced by reconstituting lethally irradiated mice with syngeneic bone marrow cells followed by a short course of therapy with the immunosuppressive agent cyclosporine A. Following cessation of cyclosporine A therapy, animals develop clinical symptoms of SGVHD: weight loss, runting, and diarrhea. While it has been suggested that T cells are responsible for the induction and effector phases of SGVHD, the role of nonspecific effector cells and cytokine mediators has yet to be examined in the disease process. Mice with SGVHD had increased levels of message for IL-12p40, IFN-gamma, and TNF-alpha in the target organs of SGVHD as compared with transplant controls and asymptomatic cyclosporine A-treated mice. Concomitant with the increase in Th1 cytokines was an enhanced cellular infiltrate in the target organs of SGVHD mice as determined by histological analysis. To directly examine the role of IL-12 in the development of SGVHD, in vivo neutralization of IL-12 was performed. Treatment of mice with Abs to IL-12 inhibited SGVHD-mediated tissue pathology and mortality. Because IL-12 has been shown to activate both T cells and NK cells to secrete IFN-gamma and to become more cytolytic, studies were initiated to ascertain which lymphocyte populations play a role in the development of murine SGVHD. Depletion of NK cells inhibited clinical symptoms of SGVHD. In contrast, T cell depletion did not alter the disease process. Therefore, these findings collectively demonstrate a role for IL-12 and NK cells in the effector phase of murine SGVHD.

Rejection of an MHC class II negative tumor following induction of murine syngeneic graft-versus-host disease.

Cyclosporin A (CsA) has been used clinically to induce graft-versus-host disease following autologous bone marrow transplantation in an attempt to destroy residual leukemia cells and reduce relapse. To analyze the antitumor potential of murine syngeneic graft-versus-host disease (SGVHD), C3H/HeN mice were lethally irradiated, reconstituted with T cell-depleted syngeneic bone marrow (ATBM) and treated with CsA for 21 days. Graft-versus-leukemia activity was assessed by challenging groups of olive oil-treated control ATBM (OO-ATBM) and CsA-treated (CsA-ATBM) mice 1 week after CsA therapy with graded doses of the syngeneic 38C13 B cell lymphoma. Following CsA treatment, up to 70% of CsA-ATBM developed SGVHD and more than 70% of the animals injected with 500 38C13 cells exhibited long-term survival (MST >80 days). In contrast, none of the OO-ATBM control mice developed SGVHD, and more than 75% of these mice died following injection of 500 38C13 tumor cells (MST = 34 days). Long-term survivors were not resistant to tumor challenge suggesting that tumor-specific immunity did not develop. Finally, class II negative 38C13 cells cultured in IL-4 or IL-10 were not inducible for MHC class II molecules, demonstrating that class II-independent antitumor mechanisms exist in SGVHD mice.

Neonatal alloimmune thrombocytopenia due to anti-HPA-1b (PLA2)(Zwb). A case report and review.

BACKGROUND: Neonatal alloimmune thrombocytopenia is a rare condition due to passively acquired maternal antibodies directed against paternal platelet antigens inherited by the infant. Only 5 cases have been reported due to antibodies against HPA-1b (PLA2) (Zwb). CASE REPORT: We report a case of neonatal alloimmune thrombocytopenia due to anti-HPA-1b in the second pregnancy of a 26-year-old Caucasian female. The male infant was treated with a 5-day course of intravenous immunoglobulin without complications. We report the HLA phenotype of the infant's mother and summarize the previous case reports due to anti-HPA-1b. CONCLUSION: Based on this case and a review of the literature, intravenous immunoglobulin as well as random donor exchange transfusion and random donor platelet transfusions are effective in the treatment of neonatal alloimmune thrombocytopenia due to anti-HPA-1b. Obvious associations between HLA alleles and sensitization to HPA-1b have not been elucidated.

Idiopathic pneumonia syndrome in mice after allogeneic bone marrow transplantation.

Pulmonary complications are a major clinical problem following allogeneic bone marrow transplantation (BMT), contributing to more than 30% of transplant-related mortalities. Idiopathic pneumonia syndrome is responsible for significant mortality among BMT patients. However, the etiology of injury to the lung parenchyma by this disease syndrome is unknown and it has been difficult to evaluate the cellular and molecular mechanisms underlying IPS in the absence of a suitable animal model. To study post-BMT lung disease during graft-versus-host disease (GVHD), we have developed a murine model that utilizes a semi-allogeneic parental --> F1 transplant strategy to induce a mild form of GVHD. Progressive inflammatory lung disease developed in animals with mild GVHD, as indicated by changes in immune cell distribution and cytokine expression in the lungs of transplanted animals. Histologic analysis of lung tissue from GVHD mice at 3 wk post-BMT showed minor immunopathologic changes compared with control mice. In contrast, lungs of GVHD mice at 12 wk displayed histopathologic hallmarks of interstitial pneumonitis, such as prominent perilumenal mononuclear cell infiltration and areas of alveolar congestion. Flow cytometric analysis of lung interstitial cells of GVHD mice revealed an increase in CD8+ T-cells at week 3, which decreased to normal levels by week 12 post-BMT. Simultaneously, the percentage of CD4+ T-cells increased progressively above normal levels and peaked at week 7 post-BMT. Analysis of cytokine mRNA expression in lung tissue indicated that steady state levels of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, interferon-gamma, and IL-12 were significantly elevated in lungs of GVHD mice at 3 wk post-BMT compared with untreated controls. Mice that were transplanted with allogeneic bone marrow alone (BMT controls) also displayed elevated expression of these cytokines, although only IL-6 was significantly higher than in untreated controls. In contrast, at 12 wk after transplantation only TNF-alpha and IL-12 levels remained elevated in GVHD mice, suggesting prolonged macrophage activation. On the basis of these findings, we conclude that allogeneic bone marrow transplantation in this mouse model causes a progressive interstitial pneumonitis, which is characterized by an acute influx of CD8+ T-cells, followed in the chronic phase by a prominent accumulation of CD4+ T-cells, and is associated with persistent production of cytokines known to activate macrophages.

Inhibition of graft-versus-host disease. Use of a T cell-controlled suicide gene.

Graft-vs-host disease (GVHD) after allogeneic bone marrow transplantation is associated with significant morbidity and mortality. T cell depletion of the marrow graft, which is currently used to prevent GVHD, has been shown to result in increased graft failure and leukemia relapse. To explore the feasibility of controlling GVHD, transgenic mice with the herpes simplex virus thymidine kinase suicide gene linked to the IL-2 promoter were used as a source of T cells to induce GVHD, which would be modulated with ganciclovir. Injection of herpes simplex virus thymidine kinase transgenic B10.A(5R) spleen cells into lethally irradiated DBA/2 mice resulted in severe acute GVHD. Treatment of the recipient mice with ganciclovir significantly inhibited GVHD-mediated weight loss and mortality and reduced the severity of inflammation in the target organs of GVHD.

Enhanced graft-versus-host disease in older recipient mice following allogeneic bone marrow transplantation.

The incidence and severity of GVHD following bone marrow transplantation increases with recipient age. The role of recipient age on the development of GVHD was analyzed in a semi-allogeneic (C57BL/6-->(C57BL/6 x DBA/2)F1) murine GVHD system. Young adult (2 months) and old (12-14 months) recipient mice were lethally irradiated and reconstituted with young adult T cell-depleted bone marrow (ATBM) or ATBM and young spleen cells. A significantly higher percentage of old vs young recipients developed lethal GVHD. Furthermore, while pre-transplant conditioning with irradiation was not required to observe increased mortality in old recipients, irradiation predisposed the older animals for a more severe course of GVHD, suggesting that GVHD occurred in old compared to young animals in the absence of pre-transplant conditioning but was exacerbated by irradiation. Histologically, the immunological responses in the GVHD target organs were more severe in the old GVHD animals. In support of this observation, increased spontaneous proliferation was observed using lymphoid cells isolated from old vs young GVHD mice. These findings demonstrate that old recipients develop a more severe course of GVHD following BMT, and may present a unique opportunity to study age-related factors in the generation of GVHD.

A cell line selected for resistance to ionizing radiation exhibits cross resistance to other genotoxic agents and a mutator phenotype for loss of heterozygosity events.

An ionizing radiation resistant derivative was obtained from the mouse P19H22 (aprt hemizygote) embryonal carcinoma cell line by repeated exposure to 137Cs gamma radiation. Ionizing radiation resistance in the 6Gy-R cell line was not correlated with a failure to undergo cell cycle arrest or a loss of the p53 response after exposure to 137Cs gamma radiation. Moreover, the cells did not display increased resistance to bleomycin, a double strand break inducing agent. However, the cells did display increased resistance to ultraviolet radiation, ethyl methanesulfonate, and 95% oxygen. A mutational analysis demonstrated a > 700 fold-fold increase in the frequency of aprt mutants for the 6Gy-R cells, but no change in the frequency of hprt or dhfr mutants. A molecular analysis suggested that the aprt mutations in the 6Gy-R cells arose by recombinational events. A possible association between radiation resistance, DNA repair, and a mutator phenotype for large-scale mutational events is discussed.

Analysis of blood T-cell cytokine expression in B-chronic lymphocytic leukaemia: evidence for increased levels of cytoplasmic IL-4 in resting and activated CD8 T cells.

The cytoplasmic cytokines of purified blood T cells (CD4/CD8) in B-CLL patients (n = 5) and controls (n = 5) were evaluated by flow cytometry. The mean levels of cytoplasmic IL-4 were significantly elevated in resting and activated B-CLL CD8 cells compared to control CD8 cells. IL-4 cytoplasmic levels were comparable for resting B-CLL and control CD4 cells but greater for B-CLL activated CD4 cells. The mean fluorescence intensity of B-CLL CD8 cytoplasmic IL-4 was 4-5-fold greater, indicating higher IL-4 density per CLL CD8 than control CD8 cells. Both CLL CD4 and CD8 cells post-activation had higher levels of cells double positive for cytoplasmic IL-4 and interferon. These data indicate that freshly isolated CD8 and CD4 blood T cells from B-CLL patients have significantly elevated (above control) levels of commitment to expression of IL-4. Since IL-4 has an important modulatory impact on CLL and normal B cells, this observation has implications regarding the biology of B-CLL.

Utility of DRB1 subtyping: a case report.

The purpose of presenting this case is to illustrate the importance of high-resolution DNA Class 2 typing when assignment of MHC antigens is of extreme importance (i.e. bone marrow transplantation); suggest this test as a substitute for the mixed lymphocyte culture (MLC), and provide evidence that DRB1 subtype mismatches may be clinically significant. Initial serological and monoclonal HLA Class 1 and low-resolution DNA-SSP Class 2 typing of a potential bone marrow transplant patient and two sisters revealed all to be HLA identical with apparent homozygosity for DRB1 x 04. High-resolution DNA-SSP Class 2 typing was then performed and revealed electrophoretic banding patterns which were not specific for any DRB1 x 04 subtype. One sister and the patient had identical patterns, while the other sister had a different pattern. Complete HLA Class 1 and high-resolution DNA-SSP Class 2 typing of the parents was performed. The mother was found to be heterozygous for Class 1 and Class 2 antigens and possess the DRB1 x 0404, 0405 subtypes. In contrast, the father was found to be homozygous for Class 1 antigens, heterozygous for Class 2 antigens and possess the DRB1 x 0404, 0407 subtypes. This led to the initial false assumption of HLA identity for the patient and her two sisters. However, assignment of haplotypes revealed one sister to be HLA identical with the patient and the other sister to be a one-haplotype five-antigen match with the patient, mismatching for one DRB1 allele. Bone-marrow transplantation was performed utilizing the latter sister, which resulted in intractable acute graft vs. host disease that resulted in the patient's demise.