Development of HSV-specific CD4+ Th1 responses and CD8+ cytotoxic T lymphocytes with antiviral activity by vaccination with the HSV-2 mutant ICP10DeltaPK.

A growth compromised herpes simplex virus type 2 (HSV-2) mutant which is deleted in the PK domain of the large subunit of ribonucleotide reductase (ICP10DeltaPK) protects from HSV-2 challenge in the mouse and guinea pig cutaneous and vaginal models and reduces the incidence and frequency of recurrent disease (Vaccine (17) (1999) 1951; Vaccine (19) (2001) 1879). The present studies were designed to identify the immune responses induced by ICP10DeltaPK and define the component responsible for protective activity. We found that ICP10DeltaPK elicits a predominant HSV-specific T helper type 1 (Th1) response, as evidenced by: (1) higher levels of HSV-specific IgG2a (Th1) than IgG1 (Th2) isotypes and (2) higher numbers of CD4+ IFN-gamma than IL-10 secreting T cells in popliteal lymph nodes. This Th1 response pattern was associated with a significant increase in the levels of IL-12 produced by dendritic cells from ICP10DeltaPK than HSV-2 immunized animals. Lymph node cells (LNCs) from ICP10DeltaPK immunized mice had significantly higher levels of HSV-2 specific cytolytic activity than LNCs from mice immunized with HSV-2 and it was mediated by CD8+ T cells. CD8+ CTL were not seen in LNCs from HSV-2 immunized mice. In adoptive transfer experiments, CD8+ T cells and, to a lower extent, CD4+ T cells from ICP10DeltaPK immunized mice inhibited HSV-2 replication, suggesting that they are involved in the protective immunity induced by ICP10DeltaPK vaccination.

A novel gene expressed in human keratinocytes with long-term in vitro growth potential is required for cell growth.

The herpes simplex virus large subunit of ribonucleotide reductase differs from its counterparts in eukaryotic and prokaryotic cells and in other viruses in that it contains a unique domain that codes for a distinct serine-threonine protein kinase that activates the Ras/MEK/MAPK mitogenic pathway and is required for virus growth. Previous studies suggested that ribonucleotide reductase protein kinase was co-opted from a cellular gene. Cellular genes similar to ribonucleotide reductase protein kinase were not cloned, however, and their function is unknown. Here we report that a novel gene (H11) that codes for a protein similar to herpes simplex virus 2 ribonucleotide reductase protein kinase, is expressed in skin tissues, cultured keratinocytes, and the keratinocyte cell line A431. The protein is phosphorylated and it associates with the plasma membrane. H11 is expressed in keratinocytes with long-term in vitro growth potential and is coexpressed with high levels of adhesion molecules involved in signal transduction, such as beta1 integrin. Antisense oligonucleotides that inhibit H11 expression inhibit DNA synthesis and keratinocyte proliferation, suggesting that H11 expression is required for cell growth.

Cellulose acetate phthalate (CAP): an 'inactive' pharmaceutical excipient with antiviral activity in the mouse model of genital herpesvirus infection.

The spread of sexually transmitted infections caused by herpes simplex virus type 2 (HSV-2) has continued unabated. At least 20% of the United States population has been infected with HSV-2 and there is a high probability of further virus transmission by asymptomatic carriers. Given the absence of effective vaccines, this indicates the need to develop prophylactic measures such as topical microbicides that have antiviral activity. Recent studies indicate that cellulose acetate phthalate (CAP), an inactive pharmaceutical excipient commonly used in the production of enteric tablets and capsules, is a broad specificity microbicide against diverse sexually transmitted pathogens. When appropriately formulated in micronized form, it inactivates various viruses, including HSV-2, in vitro. Here we show that CAP inhibits HSV-2 infection in the mouse model of genital HSV-2 infection. Pretreatment with micronized CAP formulated in a glycerol-based cream with colloidal silicone dioxide significantly reduced the proportion of HSV-2-infected mice (10% virus shedding, 0-5% lesion development and 0% fatality for CAP as compared to 84% shedding, 63% lesion development and 63% fatality in saline-treated mice). These differences were significant (P < or = 0.0002 by the test of equality of two proportions). Virus titres in the minority of mice that developed infection were similar to those in untreated mice. HSV-2 infection was not inhibited by treatment with CAP formulated with other inactive ingredients (for example povidone plus crosprovidone) instead of silicone dioxide, presumably reflecting CAP complexation/inactivation. These data suggest that properly formulated, CAP may be an efficacious agent for preventing vaginal transmission of genital herpesvirus infections.

An endogenously processed self peptide and the corresponding exogenous peptide bound to the same MHC class II molecule could be distinct ligands for TCR with different kinetic stability.

Immunization with self peptides often elicits activation of CD4+ T cells in vivo. Although such peptides have been suggested to be derived from minor self determinants or self antigens sequestered from the immune system, we found that immunization with Ealpha peptide (Ealpha52-68), a major self determinant bound to I-Ab molecules, elicits an immune response in Ealpha-transgenic C57BL/6 (Ealpha-B6) mice where Ealpha52-68 is endogenously processed and presented by I-Ab molecules in the thymus and periphery. To better understand this response, a panel of T cell hybridomas raised against exogenous Ealpha52-68 were analyzed for their reactivity to spleen cells from Ealpha-B6 mice. Some hybridomas were stimulated with Ealpha-B6 spleen cells in the absence of exogenous Ealpha52-68, whereas others were not stimulated with them. The Ealpha52-68/I-Ab complex recognized by the TCR that is expressed on the hybridoma with reactivity to Ealpha-B6 spleen cells was found to be quite stable, whereas the complex recognized by the TCR on the hybridoma specific for the exogenous Ealpha52-68 lost the stimulation activity by incubation the complex at 37 degrees C for 10 min. Stimulation experiments using extensively substituted Ealpha analogue peptides suggested that amino acid residues at positions 57, 58, 60 and 62 of Ealpha52-68 are involved in the interaction with TCR recognizing the Ealpha52-68/I-Ab complex expressed on Ealpha-B6 spleen cells. While amino acid substitutions at positions 60 and 62 also affected the recognition of TCR specific for exogenous Ealpha52-68, all or many amino acid substitutions were allowed at position 58 or 57, respectively, without impairing the TCR recognition. Taken together, these results suggest that endogenously processed self peptide and the corresponding exogenous peptide bound to the same MHC class II molecule could be distinct TCR ligands with different kinetic stability and probably with different configuration.

Highly restricted T cell repertoire shaped by a single major histocompatibility complex-peptide ligand in the presence of a single rearranged T cell receptor beta chain.

The T cell repertoire is shaped by positive and negative selection of thymocytes through the interaction of alpha/beta-T cell receptors (TCR) with self-peptides bound to self-major histocompatibility complex (MHC) molecules. However, the involvement of specific TCR-peptide contacts in positive selection remains unclear. By fixing TCR-beta chains with a single rearranged TCR-beta irrelevant to the selecting ligand, we show here that T cells selected to mature on a single MHC-peptide complex express highly restricted TCR-alpha chains in terms of Valpha usage and amino acid residue of their CDR3 loops, whereas such restriction was not observed with those selected by the same MHC with diverse sets of self-peptides including this peptide. Thus, we visualized the TCR structure required to survive positive selection directed by this single ligand. Our findings provide definitive evidence that specific recognition of self-peptides by TCR could be involved in positive selection of thymocytes.

Positive and negative CD4+ thymocyte selection by a single MHC class II/peptide ligand affected by its expression level in the thymus.

The central event in thymic selection of T cells bearing alpha beta TCRs is their interaction with self-peptides bound to self-MHC molecules. With the use of transgenic mouse lines expressing a single peptide/MHC class II complex, we show that CD4+ T cells with the preferential usage of particular TCR V(alpha)s and V(beta)s were selected to mature on this complex in lines with the lower expression, whereas such CD4+ T cells were eliminated in the thymus in a line with the relatively high expression. When a low expressing line was crossed with a high expressing line, the frequency of CD4+ T cells selected by this complex markedly decreased. Thus, these results suggest that a single peptide/MHC class II complex, being affected by its cell surface density in the thymus, can serve as both positively and negatively selecting ligand in vivo.

[Apatite-collagen complex. Preparation of a new apatite-collagen complex].

A new apatite-collagen complex was prepared in calcium beta-glycerophosphate solutions at pH 8.50. For this preparation, reconstituted type I collagen was cross-linked with phosvitin in the presence of alkaline phosphatase by use of a cross-linking agent of dimethyl suberimidate. After two weeks of immersion in daily-renewed solution of calcium beta-glycerophosphate, the complex contained apatite approximately two times the modified collagen in weight. When viewed in a scanning electron microscope, needle-like crystals precipitated densely on the collagen fibrils. However, in some portion of the complex, dot-like precipitate was observed as well. X-ray diffraction and IR analyses of the complex suggested that the apatite precipitated on the collagen fibrils was very similar to bone mineral in two aspects, crystallinity and carbonate content.

[Weibull statistical analysis of bending strength data measured either in air or in distilled water for sintered hydroxyapatite ceramics coated on the metal substrate].

To assess the effects of H2O on the fracture stress of sintered hydroxyapatite (HAP) ceramics prepared by procedures similar to those used for HAP coated on the metal substrate, four-point bending tests were carried out at 0.5 mm/min either in air (20 C, R.H. 73%) or in distilled water (37 degrees C). Then, strength data obtained were analyzed using two-parameter weibull statistics. In each condition, the bending strength data gave a good fit to single-mode weibull distribution. Weibull analysis of the data gave weibull parameter m = 7.8, sigma 0 = 26.2 MPa in air and m = 8.1, sigma 0 = 18.5 MPa in distilled water, which were calculated assuming a surface flaw model. The mean value of bending strength was 27.3 MPa in air and 18.2 MPa in distilled water. These results indicated that a corrosive environment such as H2O affects fracture stress when measured at a constant stress rate. This effect is considered to be caused by slow crack growth of cracks, occurring at a loading level lower than those at which specimens will fail. Using the weibull distribution function estimated in this study, the effects of both dimension of specimen and stress distribution in the specimen on the mean fracture stress have been predicted.

[Estimation of fatigue parameter and life-time prediction using SPT diagram for hydroxyapatite coating layer in 37 degrees C distilled water].

Lifetime and survival probability of brittle materials under given conditions of stress distribution, environment and component size can be predicted using an SPT (Strenght-Probability-Time) diagram. The SPT diagram for sintered hydroxyapatite (HAP) coated on the metal substrate via the fused glass was drawn and the safe working stress in 37 degrees C distilled water was estimated. The dynamic fatigue test carried out in 37 degrees C distilled water gave the fatigue parameter n = 19 for sintered HAP. This parameter is constant except that it depends on material and environment, and the greater the value of n, the greater the resistance to fatigue failure. For sintered HAP, therefore, the resistance to fatigue failure under this wet condition is low. To draw the SPT diagram, anchor points were taken from the fracture stress and time-to-failure data measured at 1 mm/min. The SPT diagram obtained suggested that a lifetime of 20 years with survival probability of 99.9% required the safe working stress of less than 3.2 MPa. The effects of accuracy of fatigue parameter on the uncertainty in SPT diagram were estimated.

[Self-setting apatite cement. 6. Possibility as bone substitute].

Self-setting apatite cement was investigated to evaluate its use as a possible bone substitute in the rat femur. The implant sites were recovered at intervals up 12 weeks postoperatively and investigated by the use of x-ray diffraction, contact microradiography, light and electron microscopy. By x-ray diffraction analysis, the cement placed for at least one day in the medullary canal of rats was found to be completely converted to a set phase of hydroxyapatite resembling the main inorganic phase of bone. In any specimens prepared at 1, 4, 12 weeks after implantation, no appreciable foreign body response was observed in the tissue around the set cement. At four weeks after implantation the set cement was in tight contact with the newly formed bone which appeared to involve osteocytes in lacunae and osteoblastic cells on its surface. At twelve weeks after implantation, the newly formed bone tended to grow into the interior of the set cement. With scanning electron microscopy, the newly formed bone was found to be directly deposited on the set cement. The newly formed bone consisted of fine needle-like crystals. These results strongly suggest that this cement is well tolerated by bone tissue and osteogenesis when used as a bone substitute. The advantage of the present material as a promising bone substitute is that it can be filled in surgical or traumatic bone defect as a slurry or paste.

Small angle X-ray scattering study on Lingula unguis shell.

Lingula unguis shell yields a diffuse small angle X-ray scattering which is caused mainly by the scattering from particles of apatite. In this study, the distribution of particles smaller than 500A was analyzed using the small angle X-ray scattering technique. The non-linear Guinier plot indicates that the shell contains various sizes of apatite particles ranging about 20-460A. The small granules with size about 20-160A in diameter are located in the marginal part of the shell and progressively larger granules are observed internally towards the central part. The scattering is anisotropic in the lateral and central part, where the apatite crystals highly ordered. The combined analysis of wide and small angle diffraction indicates that the particles are elongated in the c-axis direction and, furthermore, their long axes are arranged almost parallel to the direction of growth in these parts. This suggests that small and isotropic particles are formed in the early stage of mineralization and larger ellipsoidal shaped particles are formed in the later stage of mineralization.