Recombinant HA-based vaccine outperforms split and subunit vaccines in elicitation of influenza-specific CD4 T cells and CD4 T cell-dependent antibody responses in humans.

Although traditional egg-based inactivated influenza vaccines can protect against infection, there have been significant efforts to develop improved formats to overcome disadvantages of this platform. Here, we have assessed human CD4 T cell responses to a traditional egg-based influenza vaccine with recently available cell-derived vaccines and recombinant baculovirus-derived vaccines. Adults were administered either egg-derived Fluzone®, mammalian cell-derived Flucelvax® or recombinant HA (Flublok®). CD4 T cell responses to each HA protein were assessed by cytokine EliSpot and intracellular staining assays. The specificity and magnitude of antibody responses were quantified by ELISA and HAI assays. By all criteria, Flublok vaccine exhibited superior performance in eliciting both CD4 T cell responses and HA-specific antibody responses, whether measured by mean response magnitude or percent of responders. Although the mechanism(s) underlying this advantage is not yet clear, it is likely that both qualitative and quantitative features of the vaccines impact the response.

T-Cell Responses in Adults During Natural Respiratory Syncytial Virus Infection.

Background: The pathogenesis of respiratory syncytial virus (RSV) in older adults may be due to age-related T-cell immunosenescence. Thus, we evaluated CD4 and CD8 T-cell responses during RSV infection in adults across the age spectrum. Methods: Peripheral blood mononuclear cells collected during RSV infection in adults, age 26-96 years, were stimulated with live RSV and peptide pools representing F, M, NP, and G proteins and analyzed by flow cytometry. Results: There were no significant age-related differences in frequency of CD4+ T cells synthesizing interferon (IFN)γ, interleukin (IL)2, IL4, IL10, or tumor necrosis factor (TNF)α or in CD8+IFNγ+ T cells. IL4+CD4+ T-cell numbers were low, as were IL13 and IL17 responses. However, in univariate analysis, CD4 T-cell IFNγ, IL2, IL4, IL10, and TNFα responses and CD8+IFNγ+ T cells were significantly increased with more severe illness requiring hospitalization. In multivariate analysis, viral load was also associated with increased T-cell responses. Conclusions: We found no evidence of diminished RSV-specific CD4 or CD8 T-cell responses in adults infected with RSV. However, adults with severe disease seemed to have more robust CD4 and CD8 T-cell responses during infection, suggesting that disease severity may have a greater association with T-cell responses than age.

Laser welding of cast titanium and dental alloys using argon shielding.

PURPOSE: This study investigated the effect of argon gas shielding on the strengths of laser-welded cast Ti and Ti-6Al-7Nb and compared the results to those of two dental casting alloys. MATERIALS AND METHODS: Cast plates of Ti, Ti-6Al-7Nb, gold, and Co-Cr alloy were prepared. After polishing the surfaces to be welded, two plates were abutted and welded using Nd:YAG laser at a pulse duration of 10 ms, spot diameter of 1 mm, and voltage of 200 V. Five specimens were prepared for each metal by bilaterally welding them with three or five spots either with or without argon shielding. The failure load and percent elongation were measured at a crosshead speed of 1.0 mm/min. RESULTS: The factor of argon shielding significantly affected the failure load and elongation of the laser-welded specimens. The failure loads of argon-shielded laser-welded CP Ti and Ti-6Al-7Nb were greater compared with the failure loads of specimens welded without argon shielding for both three- and five-spot welding. Regardless of argon shielding, the failure loads of the laser-welded gold alloy were approximately half that of the control specimens. In contrast, the failure loads of the nonshielded laser-welded Co-Cr alloy were greater. The percent elongations positively correlated with the failure loads. CONCLUSIONS: The use of argon shielding is necessary for effective laser-welding of CP Ti and Ti-6Al-7Nb but not for gold and Co-Cr alloy.

Tensile strength and elongation of laser-welded Ti and Ti-6AL-7NB.

This study established data demonstrating the possible laser-welded strengths of cast Ti and Ti-6Al-7Nb and compared them to those of two dental-casting alloys. Cast plates of Ti, Ti-6Al-7Nb, gold, and Co-Cr alloy were prepared. After polishing the surfaces to be welded, two plates were abutted and welded using an Nd:YAG laser at a pulse duration of 10 ms, spot diameter of 1 mm, and voltage of 200 V. Five specimens were prepared for each metal by welding either three or five spots unilaterally or bilaterally. The fracture load and percent elongation were measured at a crosshead speed of 1.0 mm/min. The bilaterally welded specimens performed significantly greater than unilaterally welded specimens in both fracture load and elongation whether they were welded with three or five spots per side. The bilaterally welded Ti and Ti-6Al-7Nb specimens were nearly as strong as their corresponding control specimens, whereas the gold and Co-Cr specimens were approximately half as strong. When a large proportion of the cross-sectional area of the joint is laser welded, the strength of the laser-welded portion of the cast Ti and Ti-6Al-7Nb may approach or equal that of the nonwelded metal frameworks.

The role of antigen in the localization of naive, acutely activated, and memory CD8(+) T cells to the lung during influenza pneumonia.

The role of Ag in the recruitment and localization of naive, acutely activated, and memory CD8(+) T cells to the lung during influenza infection was explored using TCR-transgenic (Tg) mice. Naive, Thy1.2(+)CD8(+) OT-I TCR-Tg cells were primed and recruited to the lung after transfer into congenic Thy1.1(+) recipients challenged with a genetically engineered influenza virus (influenza A/WSN/33 (WSN)-OVA(I)) containing the K(b) restricted OVA(257-264) epitope (siinfekl) in the viral neuraminidase stalk. However, if the transferred animals were infected with a similar influenza virus that expressed an irrelevant K(b) epitope (WSN-PEPII), no TCR-Tg T cells were detectable in the lung, although they were easily visible in the lymphoid organs. Conversely, there were substantial numbers of OT-I cells found in the lungs of WSN-PEPII-infected mice when the animals had been previously, or were concurrently, infected with a recombinant vaccinia virus expressing OVA. Similar results were obtained with nontransgenic populations of memory CD8(+) T cells reactive to a murine gamma-herpesvirus-68 Ag. Interestingly, the primary host response to the immunodominant influenza nucleoprotein epitope was not affected by the presence of memory or recently activated OT-I T cells. Thus, although Ag is required to activate the T cells, the subsequent localization of T cells to the lung during a virus infection is a property of recently activated and memory T cells and is not necessarily driven by Ag in the lung.

Differences in body fat distribution and antioxidant status in Korean men with cardiovascular disease with or without diabetes.

BACKGROUND: Abnormal body fat distribution and reduced antioxidant status have been shown to be effective markers of risk of cardiovascular disease (CVD). OBJECTIVE: The objective of this study was to determine the differences in body fat distribution and antioxidant status in healthy men (control subjects) and in men with CVD with or without diabetes. DESIGN: An oral-glucose-tolerance test was performed and CVD patients were subdivided into groups according to the presence or absence of diabetes. Adipose tissue areas were calculated from computed tomography scans made at the L1 and L4 vertebrae. Fasting serum concentrations of lipids, testosterone, insulin-like growth factor I, antioxidants, and plasma homocysteine were determined. RESULTS: There were no significant differences in mean age, body mass index (in kg/m(2)), or blood pressure between the groups. The visceral fat area at the L1 vertebra was nonsignificantly greater in CVD patients without diabetes than in control subjects, whereas it was significantly greater in CVD patients with diabetes than in control subjects at both the L1 and L4 vertebrae. Both groups of CVD patients had higher plasma concentrations of homocysteine and lower serum insulin-like growth factor I concentrations and superoxide dismutase activities than did control subjects. Serum ss-carotene and lycopene concentrations were lowest in the CVD patients with diabetes. CONCLUSION: The concurrent presence of CVD and diabetes is associated with a greater negative effect on the risk factors typically associated with significant declines in health status.

Targeted disruption of the CP2 gene, a member of the NTF family of transcription factors.

The NTF-like family of transcription factors have been implicated in developmental regulation in organisms as diverse as Drosophila and man. The two mammalian members of this family, CP2 (LBP-1c/LSF) and LBP-1a (NF2d9), are highly related proteins sharing an overall amino acid identity of 72%. CP2, the best characterized of these factors, is a ubiquitously expressed 66-kDa protein that binds the regulatory regions of many diverse genes. Consequently, a role for CP2 has been proposed in globin gene expression, T-cell responses to mitogenic stimulation, and several other cellular processes. To elucidate the in vivo role of CP2, we have generated mice nullizygous for the CP2 allele. These animals were born in a normal Mendelian distribution and displayed no defects in growth, behavior, fertility, or development. Specifically, no perturbation of hematopoietic differentiation, globin gene expression, or immunological responses to T- and B-cell mitogenic stimulation was observed. RNA and protein analysis confirmed that the nullizygous mice expressed no full-length or truncated version of CP2. Electrophoretic mobility shift assays with nuclear extracts from multiple tissues demonstrated loss of CP2 DNA binding activity in the -/- lines. However, a slower migrating complex that was ablated with antiserum to NF2d9, the murine homologue of LBP-1a, was observed with these extracts. Furthermore, we demonstrate that recombinant LBP-1a can bind to known CP2 consensus sites and form protein complexes with previously defined heteromeric partners of CP2. These results suggest that LBP-1a/NF2d9 may compensate for loss of CP2 expression in vivo and that further analysis of the role of the NTF family of proteins requires the targeting of the NF2d9 gene.

Analysis of the virus-specific and nonspecific B cell response to a persistent B-lymphotropic gammaherpesvirus.

Respiratory challenge of mice with murine gammaherpesvirus 68 (gammaHV68) results in acute replication in respiratory epithelial cells and persistent, latent infection of B cells and macrophages. gammaHV68 elicits virus-specific Ab, and also nonspecifically activates B cells to Ab production through a CD4+ T cell-dependent process. The current analysis characterizes virus-specific and nonspecific Ab production at the single cell level and investigates the requirements and nature of the nonspecific response. Virus-specific Ab-forming cell (AFC) numbers were dwarfed by the increase in total AFC in all sites examined, indicating substantial nonspecific Ab production. Clear increases and decreases in specific and total AFC numbers occurred in the lymph nodes draining the respiratory tract and the spleen, but AFC numbers in the bone marrow (BM) increased to a plateau and remained constant. The longevity of the BM response was reflected in a sustained increase in virus-specific and total serum Ab levels. Generally, the IgG2a and IgG2b isotypes predominated. Analysis of cytokine-deficient mice, CD40 ligand-deficient mice, and radiation BM chimeras lacking MHC class II expression specifically on B cells indicated that nonspecific Ab production is independent of IL-6 or IFN-gamma, and dependent on cognate CD4+ T cell help. Several observations were consistent with polyclonal B cell activation by gammaHV68, including the induction of durable serum levels of IgG reactive with mammalian dsDNA and murine type II collagen. Our findings indicate new directions for studies of this valuable model of gamma-herpesvirus pathogenesis.

Disposition of glucosinolates and sulforaphane in humans after ingestion of steamed and fresh broccoli.

The cancer-chemopreventive effects of broccoli may be attributed, in part, to isothiocyanates (ITCs), hydrolysis products of glucosinolates. Glucosinolates are hydrolyzed to their respective ITCs by the enzyme myrosinase, which is inactivated by heat. In this study, the metabolic fate of glucosinolates after ingestion of steamed and fresh broccoli was compared in 12 male subjects in a crossover design. During each 48-hour baseline period, no foods containing glucosinolates or ITCs were allowed. The subjects then consumed 200 g of fresh or steamed broccoli; all other dietary sources of ITCs were excluded. Blood and urine samples were collected during the 24-hour period after broccoli consumption. Total ITC equivalents in broccoli and total ITC equivalents in plasma and urine were assayed by high-performance liquid chromatography as the cyclocondensation product of 1,2-benzenedithiol. The content of ITCs in fresh and steamed broccoli after myrosinase treatment was found to be virtually identical (1.1 vs. 1.0 micromol/g wet wt). The average 24-hour urinary excretion of ITC equivalents amounted to 32.3 +/- 12.7% and 10.2 +/- 5.9% of the amounts ingested for fresh and steamed broccoli, respectively. Approximately 40% of total ITC equivalents in urine, 25.8 +/- 13.9 and 6.9 +/- 2.5 micromol for fresh and steamed broccoli, respectively, occurred as the N-acetyl-L-cysteine conjugate of sulforaphane (SFN-NAC). Total ITC metabolites in plasma peaked between 0 and 8 hours, whereas urinary excretion of total ITC equivalents and SFN-NAC occurred primarily between 2 and 12 hours. Results of this study indicate that the bioavailability of ITCs from fresh broccoli is approximately three times greater than that from cooked broccoli, in which myrosinase is inactivated. Considering the cancer-chemopreventive potential of ITCs, cooking broccoli may markedly reduce its beneficial effects on health.

The distribution of porcine pancreatic beta-cells at ages 5, 12 and 24 weeks.

Islet transplantation is a potential treatment for diabetes mellitus and porcine pancreata may provide a readily available source of islets. The size, number and distribution of islets within the pancreas may influence the choice of age of donor for xenotransplantation. Samples (n = 3 per age group) from the dorsal and ventral pancreas of 5-, 12- and 24-week-old hybrid pigs were fixed in formal saline, processed in paraffin wax and stained with an avidin/biotin immunohistochemical kit for insulin, glucagon, somatostatin and pancreatic polypeptide. The arrangement of endocrine cells within the pancreata were studied and mean diameter of beta-cell groups were measured (from insulin stained sections) in 1 mm2 grid areas (n = 10 per section) and collated into groups according to size. Percentage volume density of beta-cells in relation to the whole pancreas was calculated and also the distribution of beta-cell groups, according to their size, within the total beta-cell mass. There were differences in the frequency and arrangement of endocrine cells within islets at the different ages studied. beta-Cell groups < 50 microm in diameter occupied 70 to 80% of the total beta-cell mass at 5 weeks but, as the age of the pig increased, larger cell groups were more abundant. However, the percentage volume density of beta-cells within the total pancreas did not change as the pancreas matured. This study shows that the endocrine porcine pancreas was maturing and its structure changed between the ages of 5 and 24 weeks. The relevance of these findings may have implications on the isolation and function of islets if young pigs are to be used as donors for transplantation as a treatment for diabetes mellitus.

SOCS1 deficiency causes a lymphocyte-dependent perinatal lethality.

SOCS1 is an SH2-containing protein that is primarily expressed in thymocytes in a cytokine- and T cell receptor-independent manner. SOCS1 deletion causes perinatal lethality with death by 2-3 weeks. During this period thymic changes include a loss of cellularity and a switch from predominantly CD4+ CD8+ to single positive cells. Peripheral T cells express activation antigens and proliferate to IL-2 in the absence of anti-CD3. In addition, IFNgamma is present in the serum. Reconstitution of the lymphoid lineage of JAK3-deficient mice with SOCS1-deficient stem cells recapitulates the lethality and T cell alterations. Introducing a RAG2 or IFNgamma deficiency eliminates lethality. The results demonstrate that lymphocytes are critical to SOCS1-associated perinatal lethality and implicate SOCS1 in lymphocyte differentiation or regulation.

SOCS3 is essential in the regulation of fetal liver erythropoiesis.

SOCS3 (CIS3/JAB2) is an SH2-containing protein that binds to the activation loop of Janus kinases, inhibiting kinase activity, and thereby suppressing cytokine signaling. During embryonic development, SOCS3 is highly expressed in erythroid lineage cells and is Epo independent. Transgene-mediated expression blocks fetal erythropoiesis, resulting in embryonic lethality. SOCS3 deletion results in an embryonic lethality at 12-16 days associated with marked erythrocytosis. Moreover, the in vitro proliferative capacity of progenitors is greatly increased. SOCS3-deficient fetal liver stem cells can reconstitute hematopoiesis in lethally irradiated adults, indicating that its absence does not disturb bone marrow erythropoiesis. Reconstitution of lymphoid lineages in JAK3-deficient mice also occurs normally. The results demonstrate that SOCS3 is critical in negatively regulating fetal liver hematopoiesis.

Stat5 activation is uniquely associated with cytokine signaling in peripheral T cells.

The activation and subsequent proliferation of peripheral T cells requires the engagement of the T cell and a cytokine receptor, typically the IL-2 or IL-4 receptors. Critical to understanding the regulation of peripheral T cells is the knowledge of the unique contributions of each receptor to full T cell activation and cell cycle progression. Mice deficient in Stat5a and Stat5b have demonstrated the essential role that these highly related proteins play in cell cycle progression following peripheral T cell activation. Here we demonstrate that activation of the Stat5 proteins by tyrosine phosphorylation is uniquely contributed by cytokine receptor signaling and specifically does not occur through the T cell receptor complex.

Effects of retroviral-mediated MDR1 expression on hematopoietic stem cell self-renewal and differentiation in culture.

Ex vivo expansion of hematopoietic stem cells would be useful for bone marrow transplantation and gene therapy applications. Toward this goal, we have investigated whether retrovirally-transduced murine stem cells could be expanded in culture with hematopoietic cytokines. Bone marrow cells were transduced with retroviral vectors expressing either the human multidrug resistance 1 gene (HaMDR1), a variant of human dihydrofolate reductase (HaDHFR), or both MDR1 and DHFR in an internal ribosomal entry site (IRES)-containing bicistronic vector (HaMID). Cells were then expanded for 15 days in cultures stimulated with interleukin (IL)-3, IL-6, and stem cell factor. When very low marrow volumes were injected into lethally irradiated recipient mice, long-term reconstitution with 100% donor cells was seen in all mice injected with HaMDR1- or HaMID-transduced cells. By contrast, engraftment with HaDHFR- or mock-transduced cells ranged from partial to undetectable despite injection of significantly larger marrow volumes. In addition, mice transplanted with expanded HaMDR1- or HaMID-transduced stem cells developed a myeloproliferative disorder that was characterized by an increase in abnormal peripheral blood leukocytes. These results show that MDR1-transduced stem cells can be expanded in vitro with hematopoietic cytokines, but indicate that an increased stem cell division frequency can lead to stem cell damage.

Stat5 is required for IL-2-induced cell cycle progression of peripheral T cells.

Many cytokines activate two highly homologous Stat proteins, 5a and 5b. Mice deficient in both genes lack all growth hormone and prolactin functions but retain functions associated with cytokines such as erythropoietin. Here, we demonstrate that, while lymphoid development is normal, Stat5a/b mutant peripheral T cells are profoundly deficient in proliferation and fail to undergo cell cycle progression or to express genes controlling cell cycle progression. In addition, the mice lack NK cells, develop splenomegaly, and have T cells with an activated phenotype, phenotypes seen in IL-2 receptor beta chain-deficient mice. These phenotypes are not seen in mice lacking Stat5a or Stat5b alone. The results demonstrate that the Stat5 proteins, redundantly, are essential mediators of IL-2 signaling in T cells.

The influence of collagenase solvent on the isolation of islets from 5 week old pigs: a comparison of TCM-199 and UW.

Islet transplantation is a potential treatment for diabetes, but the techniques for islet isolation are inefficient and the recovery rates for isolated islets are often low. As the solutions employed during the isolation process may affect islet yield, we have investigated the effect of collagenase solvent, and compared the effect of dissolving collagenase in TCM-199 (TCM) or University of Wisconsin (UW) solution on yield and viability of islets isolated from 5 week old pigs. Pancreata were transported to the laboratory in UW solution, and the islets isolated using a manual method of collagenase digestion. The optimum concentration of collagenase which would liberate the maximum number of islets was determined for each solvent, and then the yield and viability of islets isolated using collagenase in TCM and UW were compared. It was found that, when UW was used as collagenase solvent, a higher concentration of collagenase was required to liberate the maximum number of islets. Comparative experiments revealed that although the total number of isolated islets was greater using UW as the solvent, the number of islet equivalents was similar in both preparations. More than 90% of the cells in both preparations excluded trypan blue, although according to a scoring system, preparations isolated using UW showed greater viability. The stimulation indices in response to glucose and theophylline were similar for both preparations, but islets isolated using UW generally responded with a lower but more sustained insulin release. In conclusion, there was no difference between the total amount of islet tissue isolated using TCM or UW as solvent for collagenase. The preparations isolated using UW were more fragmented, but exhibited superior viability.

Traditional Chinese medicine in orthopaedic nursing.

Increasing popularity of traditional Chinese medicine (TCM) by the American public warrants an examination of its role in all of nursing. Many people seeking alternative treatments do so out of frustration with the inability of Western medicine to help with chronic illnesses. Thus, Western practitioners need a basic understanding of these alternative modalities to help their clients make informed choices about their health care. This article describes the underlying theory of TCM, assessments, diagnostics, and treatments found in TCM, as well as uses of TCM in orthopaedic nursing. Also included are research considerations as well as precautions in the use of TCM.

Thymic lymphoproliferative disease after successful correction of CD40 ligand deficiency by gene transfer in mice.

Inherited deficiency of the CD40 ligand (X-linked hyper-IgM syndrome) is characterized by failure of immunoglobulin isotype switching and severe defects of cell-mediated immunity. To test the potential for gene transfer therapy to correct this disorder, we transduced murine bone marrow or thymic cells with a retroviral vector containing the cDNA for the murine CD40 ligand (CD40L) and injected them into CD40L-/- mice. Even low-level, constitutive expression of the transgene stimulated humoral and cellular immune functions in these mice. With extended follow-up, however, 12 of 19 treated mice developed T-lymphoproliferative disorders, ranging from polyclonal increases of lymphoblasts to overt monoclonal T-lymphoblastic lymphomas that involved multiple organs. Our findings show that constitutive (rather than tightly regulated), low-level expression of CD40L can produce abnormal proliferative responses in developing T lymphocytes, apparently through aberrant interaction between CD40L+ and TCRalphabeta+CD40+ thymocytes. Current methods of gene therapy may prove inappropriate for disorders involving highly regulated genes in essential positions in proliferative cascades.

Longitudinal analysis of the acute Sendai virus-specific CD4+ T cell response and memory.

The development and persistence of Sendai virus-specific CD4+ T cell memory has been analyzed following respiratory infection of C57BL/6J mice by determining the prevalence of IL-2-producing Th cell precursors (Thp). Frequencies as high as 1:40 virus-specific CD4+ T cells were found in the regional lymph nodes and spleen during the acute phase of the host response and persisted at levels > or =1:500 for 2 to 3 mo. Thereafter, these CD4+ T cells tended to distribute more to the spleen than to the lymph nodes, a pattern that persisted for the life of the animals. From 3 to 12 mo after infection, virus-specific Thp were always detectable, although the numbers were diminished relative to those measured during the acute phase. Thereafter, however, in both contemporary and cumulative assays, there was a progressive increase in both the frequency and number of Thp. These increases were especially apparent for mice more than 2 years of age. This may reflect enrichment of the CD4+CD44high memory set due to the gradual diminution of the naive CD4+CD62LhighCD44low component. Analysis of DNA staining profiles for the CD4+ T cells showed high levels of cycling for the acute phase of the response, whereas the rate of T cell turnover measured for the CD4+CD44high population by bromodeoxyuridine incorporation indicated a pattern of stable, continuing proliferation throughout life. Virus-specific CD4+ T cell memory resulting from a single exposure to a readily eliminated RNA virus is thus maintained indefinitely in laboratory mice.