The effects of teriparatide on acceleration of bone healing following atypical femoral fracture: comparison between daily and weekly administration.

We compared the effectiveness of promoting bone healing between two teriparatide preparations for atypical femoral fracture (AFF). A total of 45 AFFs were included in this study, and we compared the duration of bone union. Teriparatide administered by daily injection enhanced bone union more than weekly administration in complete AFFs. INTRODUCTION: The efficacy of teriparatide for atypical femoral fracture (AFF) has been recently reported. Although two different teriparatide preparations can be used to treat osteoporosis in Japan, daily or weekly injection, all previous reports on the effectiveness of teriparatide for AFF only examined daily injection formulations. Therefore, we compared the promotion of bone healing between the two teriparatide preparations for AFF. METHODS: A total of 45 consecutive AFFs in 43 Japanese patients were included in this study. They received either a daily 20-µg teriparatide injection (daily group; n = 32) or a once-a-week 56.5-µg teriparatide injection (weekly group; n = 13). We compared the clinical background and duration of bone union between these two groups. RESULTS: When all patents were included, the fracture healing time was not significantly different between the two groups. Only patients with complete AFFs had significantly fewer daily bisphosphonate or denosumab injections than the weekly group (P < 0.05). The fracture healing time in the daily group (6.1 ± 4.1 months) was significantly shorter than that in the weekly group (10.1 ± 4.2 months) (P < 0.05). Even if the influence of bisphosphonate or denosumab usage was excluded, a similar significant difference was observed in the fracture healing time (P < 0.05). There was no significant difference between the two groups among patients with incomplete AFFs. CONCLUSIONS: Daily teriparatide injections enhance bone union more than weekly injections in complete AFF patients.

ACTN3 R577X genotype is associated with sprinting in elite Japanese athletes.

The ACTN3 R577X genotype has been found to associate with sprint/power phenotypes in all elite athlete cohorts investigated. This association has not been extensively studied in elite Asian athletes. The present study was undertaken to investigate the association between the ACTN3 R577X genotype and elite Japanese track and field athlete status. 299 elite Japanese track and field athletes (134 sprint/power athletes; 165 endurance/middle-power athletes) and 649 Japanese controls were genotyped for the ACTN3 R577X polymorphism. All athletes were of national or international level. Sprint/power athletes showed a higher frequency of RR + RX genotype than controls (111/134 [82.8%] vs. 478/649 [73.7%], P = 0.025 under the R-dominant model), while there was no significant difference between endurance/middle-power athletes and controls (126/165 [76.4%] vs. 478/649 [73.7%], P = 0.48 under the R-dominant model). Sprinters with the RR + RX genotype had significantly faster personal best times for the 100 m than those with XX genotype (10.42 ± 0.05 s vs. 10.64 ± 0.09 s, P = 0.042); no such association was found in the 400 m sprinters (47.02 ± 0.36 s vs. 47.56 ± 0.99 s, P = 0.62). ACTN3 R577X genotype is associated with sprint/power performance in elite Japanese track and field athletes, especially short sprint performance.

Combined treatment of alendronate and low-intensity pulsed ultrasound (LIPUS) increases bone mineral density at the cancellous bone osteotomy site in aged rats: a preliminary study.

INTRODUCTION: During fracture healing, alendronate encourages callus volume by inhibiting bone resorption, whereas low-intensity pulsed ultrasound (LIPUS) enhances bone regeneration by promoting an anabolic response. METHODS: In the present study, 9-month-old Sprague-Dawley rats, with a unilateral proximal tibial osteotomy, were treated with alendronate (daily, 1 µg/kg) plus sham-LIPUS (n = 14), saline plus LIPUS (20 min/day) (n = 18), alendronate plus LIPUS (n = 16), or saline plus sham-LIPUS as a control (n = 13) for 4 weeks. The rats were then examined for changes in bone mineral density (BMD) during metaphyseal bone repair. RESULTS: The combined therapy significantly increased BMD at the osteotomy site at 4 weeks (p < 0.001) compared with the control, without affecting the contralateral, non-osteotomized tibia. Both alendronate and LIPUS alone also exerted a positive, albeit less, effect on BMD in the affected limb (p < 0.001 and p = 0.006, respectively). CONCLUSION: Alendronate and LIPUS cooperate to enhance BMD during metaphyseal bone healing.

Amino-terminal fragment of urokinase-type plasminogen activator inhibits HIV-1 replication.

CD8+ T lymphocytes have been shown to produce unidentified soluble factors active in suppressing HIV-1 replication. In this study, we purified an HIV-1 suppressing activity from the culture supernatant of an immortalized CD8+ T cell clone, derived from an HIV-1 infected long-term nonprogressor, and identified this activity as the amino-terminal fragment (ATF) of urokinase-type plasminogen activator (uPA). ATF is catalytically inactive, but suppresses the release of viral particles from the HIV-1 infected cell lines via binding to its receptor CD87. In contrast, cell proliferation and the secretion of an HIV-1 LTR driven reporter gene product were not affected by ATF. These findings suggest that ATF may inhibit the assembly and budding of HIV-1, which provides a novel therapeutic strategy for AIDS.

Seronegative HIV-2 carriers in India.

The discordant cases of seronegative, but culture and proviral HIV-2 DNA positive were found in Mumbai, India. This was corroborated by the successful isolation of HIV-2-RNA in culture medium, HIV-2 cDNA sequence determination and the detection of the antigen. The sequence of the isolated HIV-2 genomic RNA does not seem to be altered to the extent that the change will alter antibody binding. Furthermore, antibody from the same individual (even at 8 months from initial sampling) from whom HIV-2 was isolated did not react with the antigen of this strain. Those evidences imply that extremely low or non-production of the antibody may be due to suboptimal immune stimulation due to extremely slow HIV-2 replication. This low virus-load may be responsible for the negative antibody results in the HIV-2 carriers.

PIP: This paper describes the characteristics of HIV-2 seropositive and seronegative cases in Mumbai, India, and characterizes the differences between HIV-1 and HIV-2. More than 200 outpatients considered to be at high risk of HIV infection were screened for HIV-1 and HIV-2 antibody and proviral DNA. The study found 11 cases that were discordant for antibody test and HIV proviral DNA (i.e., negative for anti-HIV but positive for HIV-2 proviral DNA). The presence of this provirus was further corroborated by the detection of HIV-2 RNA in the culture medium upon HIV isolation, HIV-2 cDNA sequencing, and antigen detection. The sequence of the isolated HIV-2 genomic RNA did not seem to be altered to the extent that the change would affect antibody binding. Moreover, antibody from the same person in whom HIV-2 was detected did not react with the antigen of this strain even 8 months after the initial sampling. These findings indicate that extremely low production or non-production of the antibody may be brought about by suboptimal immune stimulation due to very low HIV-2 replication speed. This low virus load may account for the negative antibody results in the HIV-2 carriers in India.

Conservation of the central proline-rich (PxxP) motifs of human immunodeficiency virus type 1 Nef protein during the disease progression in two hemophiliac patients.

The nef gene is considered to play a crucial role in the development of acquired immunodeficiency syndrome (AIDS). In this study, we analyzed the sequence of nef quasispecies obtained from replication-competent HIV-1 isolates from two Japanese hemophiliac patients infected with HIV-1. At least 10 nef clones were isolated at each time point and a total of 75 individual nef quasispecies were sequenced. We observed a gradual increase in genetic diversity of the nef gene over time. Among the various functional regions of Nef protein, myristoylation site and the central PXXP (SH3 ligand) motifs were well conserved. The scattered regions responsible for downregulation of CD4 and class I MHC were also conserved. These data suggest that these functions of Nef may be involved throughout the disease process.

Difference in susceptibility to CC-chemokines among HIV-1 isolates.

In this study, we examined the difference in susceptibility to anti-HIV activity of the CC-chemokines (RANTES, MIP-1 alpha and MIP-1 beta) among HIV-1 isolates and analysed its relation with phenotype (syncytium inducibility) and V3 domain of gp120 of the HIV-1 isolates. Of 11 cases tested in endogenous assay, at a concentration of 200 ng/ml, RANTES, MIP-1 alpha, and MIP-1 beta showed more than 80% suppression of HIV-1 replication in 10, 8, and 7 cases, respectively. HIV-1 isolates sensitive to more than one CC-chemokine showed non-syncytium-inducing phenotype, whereas HIV-1 isolates resistant to all of the 3 CC-chemokines showed syncytium-inducing phenotype. HIV-1 isolates resistant to all of the 3 CC-chemokines contained more positively charged amino acid residues in the V3 domain of the gp120. These results indicated that utilization of the CC-chemokine receptors as co-receptors for virus entry could vary among HIV-1 isolates.

Slight difference in primary amino acid sequence of p17 matrix protein of HIV-1 exerts profound influence on its antigenicity.

HIV-1 p17 antigen has been studied for its biological significance in vitro as well as its immunological roles in vivo. By immunological approach of antibody-binding to HIV-1 p17 antigens of several subtypes in combination with computerized analysis of those tertial structures, it became evident that, irrelevant of similarity of linear amino acid sequence of different HIV-1 subtypes, a few amino acid substitutions close to or distant from specified epitope(s) affected their tertial structure resulting in change in ability of its binding to selected antibody. ELISA employing two monoclonal antibodies, A144 and C415, could detect p17 of subtypes A and B, but not of subtypes C, D, and E. Since the epitope site corresponding to A144 has been reported to be important for biological activity of p17 of HIV-1, change in tertial structure around this epitope may explain some difference in biology of HIV-1, such as infectivity of subtypes B and E.

Interleukin-9 receptor alpha chain mRNA formation in CD8+ T cells producing anti-human immunodeficiency virus type 1 substance(s).

A search for gene(s) associated with anti-human immunodeficiency virus type 1 (HIV-1) activity of CD8+ T cells was attempted using molecular cloning and the relation between the anti-HIV activity of CD8+ T cells and the interleukin-9 receptor alpha chain (IL-9R-alpha) mRNA expression from the cDNA clones obtained was examined. The anti-HIV-1 activity of CD8+ T cell culture supernatants was assessed by measuring the level of HIV-1 replication of a CD4+ T cell line transfected with an infectious HIV-1 DNA clone. IL-9R-alpha mRNA was assayed by reverse transcriptase-polymerase chain reaction (RT-PCR). Of 5 cases showing high level of anti-HIV-1 activity (more than 80% suppression of HIV-1 replication), the mRNA was detected in 4 cases. Of 10 cases showing low level of anti-HIV-1 activity (less than 80% suppression of HIV-1 replication), the mRNA was detected in one case. Soluble recombinant human IL-9 receptor (rhIL-9sR) did not suppress HIV-1 replication at a concentration of 1 microgram/ml. These data suggest that the IL-9R-alpha mRNA formation in CD8+ T cells may correlate with and play some role in the anti-HIV-1 activity of CD8+ T cells from HIV-1-infected individuals.

Genetic subtypes of HIV-1 in the Philippines.

OBJECTIVES: To determine the genetic variability of HIV-1 amongst infected Filipinos and to analyze phylogenetic relationships, temporal introductions and transmission dynamics of identified variants. METHODS: Polymerase chain reaction amplification and direct sequencing of a 204 base-pair fragment of the env C2-V3 region from uncultured peripheral blood mononuclear cells obtained from 51 HIV-1-positive Filipinos infected from 1987 to mid-1996. Evolutionary distance and phylogenetic relationships among the DNA sequences were estimated. RESULTS: The 51 Philippine strains were classified into five env V3 subtypes, namely subtype B (n = 37), subtype E (n = 8), subtype A (n = 3), subtype C (n = 2) and subtype D (n = 1). The overall env nucleotide divergence ranged from 11.7 to 32.2%. The nucleotide variation appeared to be random and no temporal ordering was observed. The variation of the sequences at the tip of the V3 loop was very broad. Subtypes B and C isolates did not show close genetic relationship to other Asian variants. Only three of the subtype E strains had close affinity to known Asian sequences. The majority (94%) of the subjects acquired the infection by sexual transmission. About two-thirds were presumably infected outside the Philippines, whereas the remaining were infected indigenously. Information was limited to allow segregation of the identified subtypes by mode of transmission or risk groups. CONCLUSION: Our findings demonstrate the presence of multiple genetic subtypes of HIV-1 in the Philippines. The apparent geographic range of previously reported genotypes in South and South-east Asia was extended and has obvious implications for env-based antiviral interventions.

Difference of virus populations in HIV carriers in relation to AZT treatment.

To analyse the appearance of AZT-resistant HIV in HIV carriers after AZT treatment and compare the mutations responsible for resistance employing cloned HIV DNA derived from provirus and free virions in plasma, serial blood specimens were taken before and after AZT treatment. RNA in virions in plasma, proviral DNA and RNA from virus isolates by coculture of PBMCs of HIV carriers and healthy blood donors were cloned and sequenced. DNA clones were compared for their nucleotide sequences responsible for AZT resistance. AZT resistance was acquired as early as 2 months after the start of the treatment and follow-up study was performed for 16 months of the treatment. Population of DNA clones was different according to the origin of the DNA or RNA, which indicated that the provirus population in PBMC was different from that in virions in plasma. These data demonstrated the possibility of selective activation of provirus or activation of provirus in organs other than peripheral blood, although the number of cases was small.

Suppression of HIV replication in vitro by CD8+ T-cells from HIV-infected and HIV-seronegative individuals.

The inhibitory effect of CD8+ T-cells from HIV-infected or HIV-seronegative individuals on HIV replication in the naturally-infected CD4+ T-cells in vitro was examined. Not only autologous CD8+ T-cells from HIV-infected individuals but also allogeneic CD8+ T-cells from HIV-seronegative individuals prevented or delayed HIV replication, even in transwell cocultures using a semi-permeable 0.45 micron filter. The level of the inhibitory effect of allogeneic CD8+ T-cells from the HIV-seronegative individuals on the HIV replication was varied among CD4+ T-cells obtained from HIV-infected individuals used. The results suggested that CD8+ T-cells from HIV-seronegative individuals as well as HIV-infected individuals could produce some cytokine(s) which suppress HIV replication in vitro. The sensitivity to the cytokine(s) might be variable among HIV strains, depending on differences in the nucleotide sequence of different HIV-1 strains. Further studies of control of HIV replication by CD8+ anti-HIV cytokine(s) should provide new strategies for the therapy of HIV infection.

Contribution to the regulation of virus replication in cells latently infected with human immunodeficiency virus 1.

Monocytes/macrophages have been known to play an important role in the initiation and propagation of human immunodeficiency virus 1 (HIV-1) infection. To analyze the function of these cells during the clinical asymptomatic period of infection, we examined the effect of murine peritoneal macrophages and human peripheral blood macrophages on two cell lines latently infected with HIV-1, a promonocytic cell line, U1, and a T-cell line, ACH-2. Monokines of the murine peritoneal macrophages induced significant viral expression in U1, but not in ACH-2 cells. Experiments employing transient transfection of U937 and CEM cells with HIV long terminal repeat (LTR)-chloramphenicol acetyl transferase (CAT) plasmids indicated that the effect of these monokines was due to specific activation of the HIV LTR. In contrast, supernatants of human macrophages induced viral expression in both ACH-2 and U1 cells. These results suggest that several monokines are active in regulating the transition from the clinical asymptomatic period of HIV infection to progression to acquired immunodeficiency syndrome (AIDS).

Genotypes of Japanese encephalitis virus isolated in three states in Malaysia.

Two hundred forty nucleotides from the pre-membrane gene region of 12 Japanese encephalitis virus (JEV) strains isolated from three different regions of Malaysia from 1993 to 1994 were sequenced and compared with each other and with the JEV strains from different geographic areas in Asia. These 12 Malaysian isolates were classified into two genotypes. The four JEV strains isolated from Sarawak in 1994 and the four JEV strains isolated from Sepang, Selangor in 1993 were classified into one genotype that included earlier isolated strains from Malaysia (JE-827 from Sarawak in 1968 and WTP/70/22 from Kuala Lumpur in 1970). The four JEV strains from Ipoh, Perak in 1994 were classified into another genotype that included JEV strains isolated from northern Thailand and Cambodia. In an earlier report, 10 JEV strains from Sabak Bernam, Selangor in 1992 were classified into the largest genotype that included strains isolated in temperate regions such as Japan, China, and Taiwan. The data indicate that at least three genotypes of JEV have been circulating in Malaysia.

Neutralization of human immunodeficiency virus type 1 (HIV-1) with antibody from carriers' plasma against HIV-1 protein p17.

It was investigated whether human antibody against HIV-1 protein p17 (anti-p17) in HIV carriers' plasma has the ability to neutralize the infectivity of HIV. By the pretreatment of HIV-1 with anti-p17 from HIV carriers, progeny HIV-1 production from cells infected with virus pretreated with anti-p17 was suppressed and/or delayed. The neutralizing activity of anti-p17 was decreased in the presence of recombinant p17. The latter obviously masked the neutralizing activity of anti-p17. The relevant epitope(s) on p17 is located apparently on the surface of HIV virions and the binding of anti-p17 to p17 impairs the infectivity of HIV. This implies that anti-p17, if stably present in HIV carriers' plasma, may also play an important role in reducing the infectivity of HIV-1 in vivo.

Prevalence of blood-borne viruses among intravenous drug users and alcoholics in Hiroshima, Japan.

We investigated the prevalence of human immunodeficiency viruses-1 and 2 (HIV-1 and HIV-2), human T-lymphotropic virus type I and II, hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis D virus among intravenous drug users (IVDU) in Hiroshima, Japan, where little is known about their present levels. From June to December 1993, serum samples were collected from 47 IVDU and 98 alcoholics in Hiroshima, Japan, and examined for markers of virus infection. The prevalence of antibody to HCV (anti-HCV) and/or HCV-RNA was significantly higher in IVDU than alcoholics (74.5% vs 20.4%, 44.7% vs 10.2% respectively, P < 0.001). In contrast, the prevalence of antibody to hepatitis B surface antigen and/or core antigen (anti-HBs and/or anti-HBc) showed no significant difference between the 2 groups (57.4% vs 66.3%). HIV-1 infection was found in one (2.1%) IVDU and genome analysis indicated that it was subtype B according to Myers' classification. Thus, an extremely low level of HIV infection and a high level of HCV infection was found in IVDU. Careful follow-up of this group is thought to be needed to minimize an outbreak of HIV-1 infection in Japan.

A newly developed immunofluorescence assay for simultaneous detection of antibodies to human immunodeficiency virus type 1 and type 2.

Immunofluorescence assays (IFA) that simultaneously distinguish between antibodies against closely related human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) infections have not been readily available. Serum specimens from 95 HIV-1-infected, 26 HIV-2-infected and 3 HIV-1/HIV-2 dually infected individuals and 106 seronegative blood donors were evaluated for the ability to serologically discriminate HIV-1 and HIV-2 infections by means of IFA employing three types of cells whose morphology varied within one field of microscopy. Mixtures of HIV-1-infected, HIV-2-infected and uninfected cells were used in the present study. In consequence, all serum specimens from individuals infected with HIV were confirmed to contain antibodies to HIV-1 and/or HIV-2. None of the sera from the blood donors were positive. Serum specimens from HIV-1-infected or HIV-2-infected individuals were diagnosed as single infection with HIV-1 (85/95) and HIV-2 (22/26), respectively, by this new assay. Although another 14 (10/95 and 4/26) were shown to be seropositive for both HIV-1-infected and HIV-2-infected cells, these results suggest that this assay is potentially simple and useful for screening and confirming both HIV-1 and HIV-2 infections simultaneously.

HIV-1 variants in South and South-East Asia.

HIV spread in South and South-East Asia is most alarming, and genetic variability of HIV-1 is an important consideration in vaccine development. In this study, we examined the third variable (V3) region of env gene of HIV-1 variants prevalent in Thailand, Malaysia, India, and the Philippines. By phylogenetic tree analyses, an HIV-1 variant from an injecting drug user (IDU) in Thailand belonged to subtype B, and HIV-1 variants from 2 IDUs in Malaysia were classified into 2 subtypes, B and E. One HIV-1 variant from a male homosexual in the Philippines belonged to subtype B. Out of 8 HIV-1 variants from sexually transmitted disease patients in India, 7 belonged to subtype C, and one to subtype A. Although the total number of individuals examined in this study was limited, 4 HIV-1 subtypes were found in South and South-East Asia and large international movements of HIV-1-infected individuals in this region could induce global dissemination of these HIV-1 variants.