Medical negligence and the law.

After the Consumer Protection Act, 1986, came into effect, a number of patients have filed cases against doctors. This article presents a summary of legal decisions related to medical negligence: what constitutes negligence in civil and criminal law, and what is required to prove it.

Emergence of a distinct pattern of viral mutations in chimpanzees infected with a homogeneous inoculum of hepatitis C virus.

BACKGROUND & AIMS: Prospective, long-term study of viral evolution and immunologic responses in chimpanzees infected with a homogeneous hepatitis C virus (HCV) population is crucial in understanding the pathogenesis of HCV-host interactions. METHODS: A molecular clone was constructed of HCV genotype 1b and RNA transcribed from this clone inoculated intrahepatically into chimpanzee X0142. Serum was taken from X0142 at week 2 and inoculated intravenously into a second chimpanzee (X0234). Detailed virologic, serologic, and immunologic analyses of these 2 chimpanzees were performed. RESULTS: Both chimpanzees developed persistent viremia, with titers of 10(3) to 10(5) genomes/mL, for 80 weeks (X0142) and 55 weeks (X0234) of follow-up. A late antibody response against the nonstructural proteins and a weak, transient T-helper proliferative response were detected in both animals. In X0142, 25 mutations emerged in the virus population by week 78 and 15 in X0234 by week 35. A relatively large proportion of mutations affecting protein sequences appeared in the NS5A gene (33% in X0142 and X0234 combined), and 5 mutations were common to both chimpanzees. CONCLUSIONS: In this long-term study of the molecular evolution of HCV genotype 1b from a cloned source, the appearance of a distinct pattern of mutations is suggestive of an adaptive response of HCV in vivo. In addition, a limited virus-specific immunity may contribute to HCV persistence.

Transmission of HCV to a chimpanzee using virus particles produced in an RNA-transfected HepG2 cell culture.

It was demonstrated previously that HepG2 cells produce negative strand RNA and virus-like particles after transfection with RNA transcribed from a full-length hepatitis C virus (HCV) cDNA clone [Dash et al. (1997) American Journal of Pathology, 151:363-373]. To determine in vivo infectivity of these in vitro synthesized viral particles, a chimpanzee was inoculated intravenously with HCV derived from HepG2 cells. The infected chimpanzee was examined serially for elevation of liver enzymes, for the presence of HCV RNA in the serum by reverse transcription nested polymerase chain reaction (RT-PCR), anti-HCV antibodies in the serum, and inflammation in the liver. The chimpanzee developed elevated levels of liver enzymes after the second week, but the levels fluctuated over a 10-week period. HCV RNA was detected in the serum of the chimpanzee at the second, seventh and ninth weeks after inoculation, and remained positive up to 25 weeks. Liver biopsies at Weeks 18 and 19 revealed of mild inflammation. Nucleotide sequence analysis of HCV recovered from the infected chimpanzee at the second and ninth weeks showed 100% sequence homology with the clone used for transfection studies. Serum anti-HCV antibodies were not detected by EIA during the 25 weeks follow-up period. These results suggest that intravenous administration of the virus-like particles derived from RNA-transfected HepG2 cells are infectious, and therefore, the pMO9.6-T7 clone is an infectious clone. These results provide new information that in vitro synthesized HCV particles produced from full-length HCV clone can cause infection in a chimpanzee. This study will facilitate the use of innovative approaches to the study of assembly of HCV particles and mechanisms of virus infectivity in cell culture.

A chimpanzee rhadinovirus sequence related to Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8: increased detection after HIV-1 infection in the absence of disease.

OBJECTIVE: To look for a virus related to Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8) in chimpanzees and to investigate phylogenetic and biological similarities to KSHV. METHODS: Peripheral blood mononuclear cell (PBMC) DNA samples from chimpanzees (Pan troglodytes troglodytes) were screened with newly designed consensus oligonucleotide primers for the DNA polymerase gene of KSHV-related gamma2-herpesviruses (rhadinoviruses). Samples from HIV-1-infected and -uninfected chimpanzees were screened with virus-specific primers. Antibodies to KSHV structural and latent antigens were measured by immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and Western blot. RESULTS: We identified 972 base pairs (bp) of a new viral DNA polymerase sequence with 81.6% (nucleotides) and 93.2% (protein) identity to that of KSHV/HHV8. It was detected in 15/37 (41%) animals experimentally infected with HIV-1, but only in one out of 30 uninfected animals (P<0.001). Antibodies were found by immunofluorescence to structural, but not latent, KSHV antigens in nearly all HIV-1-infected and uninfected animals. CONCLUSION: Like man and two other Old World primate species, chimpanzees harbour a virus closely related to KSHV/HHV8, termed Pan troglodytes rhadinovirus-1 (PtRV-1). Like KSHV, PtRV-1 is more easily detected by polymerase chain reaction (PCR) in the PBMC of HIV-1-infected than of HIV-1-uninfected individuals, suggesting increased viral load. Despite the close phylogenetic relationship and biological similarities between KSHV and PtRV-1, Kaposi's sarcoma (KS) has not been reported in HIV-1-infected chimpanzees. PtRV-1 may lack some of the pathogenic determinants of KSHV, or humans and chimpanzees may differ in how they control the infection with their respective rhadinoviruses.

Evolution of human and non-human primate CC chemokine receptor 5 gene and mRNA. Potential roles for haplotype and mRNA diversity, differential haplotype-specific transcriptional activity, and altered transcription factor binding to polymorphic nucleotides in the pathogenesis of HIV-1 and simian immunodeficiency virus.

Polymorphisms in CC chemokine receptor 5 (CCR5), the major coreceptor of human immunodeficiency virus 1 (HIV-1) and simian immunodeficiency virus (SIV), have a major influence on HIV-1 transmission and disease progression. The effects of these polymorphisms may, in part, account for the differential pathogenesis of HIV-1 (immunosuppression) and SIV (natural resistance) in humans and non-human primates, respectively. Thus, understanding the genetic basis underlying species-specific responses to HIV-1 and SIV could reveal new anti-HIV-1 therapeutic strategies for humans. To this end, we compared CCR5 structure/evolution and regulation among humans, apes, Old World Monkeys, and New World Monkeys. The evolution of the CCR5 cis-regulatory region versus the open reading frame as well as among different domains of the open reading frame differed from one another. CCR5 cis-regulatory region sequence variation in humans was substantially higher than anticipated. Based on this variation, CCR5 haplotypes could be organized into seven evolutionarily distinct human haplogroups (HH) that we designated HHA, -B, -C, -D, -E, -F, and -G. HHA haplotypes were defined as ancestral to all other haplotypes by comparison to the CCR5 haplotypes of non-human primates. Different human and non-human primate CCR5 haplotypes were associated with differential transcriptional regulation, and various polymorphisms resulted in modified DNA-nuclear protein interactions, including altered binding of members of the NF-kappaB family of transcription factors. We identified novel CCR5 untranslated mRNA sequences that were conserved in human and non-human primates. In some primates, mutations at exon-intron boundaries caused loss of expression of selected CCR5 mRNA isoforms or production of novel mRNA isoforms. Collectively, these findings suggest that the response to HIV-1 and SIV infection in primates may have been driven, in part, by evolution of the elements controlling CCR5 transcription and translation.

A humanized anti--4-1BB monoclonal antibody suppresses antigen-induced humoral immune response in nonhuman primates.

The interaction of 4-1BB and its ligand plays an important role in the regulation of T-cell-mediated immune responses. In this study, the authors examined the effect of a humanized anti--4-1BB monoclonal antibody (H4B4) on ovalbumin-induced immune responses in baboons. Previously, a mouse monoclonal antibody, 4B4 against the human 4-1BB molecule, was generated and characterized. Based on this antibody, a humanized version of 4B4 monoclonal antibody was constructed and the resultant antibody, H4B4, showed full recovery of the binding activity of the original antibody 4B4: a 1.5-fold increase in affinity for 4-1BB. In addition, H4B4 mediated antibody-dependent cellular cytotoxicity of activated human peripheral blood T cells and CEM cells in a dose-dependent manner. Weekly administration of H4B4 at doses of 1 or 4 mg/kg could suppress immunoglobulin G production against ovalbumin. This was not a result of the overall immune suppression, because the numbers of B and T cells and the total immunoglobulin G production were not altered during treatment with H4B4. These findings suggest that treatment with H4B4 may be a valid therapeutic approach to control unwanted immune responses in persons with autoimmune diseases.

A fast iterative nearest point algorithm for support vector machine classifier design.

In this paper we give a new fast iterative algorithm for support vector machine (SVM) classifier design. The basic problem treated is one that does not allow classification violations. The problem is converted to a problem of computing the nearest point between two convex polytopes. The suitability of two classical nearest point algorithms, due to Gilbert, and Mitchell et al., is studied. Ideas from both these algorithms are combined and modified to derive our fast algorithm. For problems which require classification violations to be allowed, the violations are quadratically penalized and an idea due to Cortes and Vapnik and Friess is used to convert it to a problem in which there are no classification violations. Comparative computational evaluation of our algorithm against powerful SVM methods such as Platt's sequential minimal optimization shows that our algorithm is very competitive.

Improvements to the SMO algorithm for SVM regression.

This paper points out an important source of inefficiency in Smola and Schölkopf's sequential minimal optimization (SMO) algorithm for support vector machine (SVM) regression that is caused by the use of a single threshold value. Using clues from the KKT conditions for the dual problem, two threshold parameters are employed to derive modifications of SMO for regression. These modified algorithms perform significantly faster than the original SMO on the datasets tried.

Postexposure immunoprophylaxis of primary isolates by an antibody to HIV receptor complex.

mAb B4 is a monoclonal antibody directed against HIV receptor complex. The antibody had broad neutralizing activity against HIV and provided postexposure prophylaxis to hu-peripheral blood leukocyte (PBL)-severe combined immunodeficient mice and chimpanzees. B4 recognized a complex receptor site for HIV on the T cell surface that includes CD4 and also may be influenced by interaction with HIV coreceptors. mAb B4 preferentially neutralized primary HIV-1 isolates compared with T cell line-adapted strains, including syncytium-inducing and non-syncytium-inducing phenotypes, representatives from HIV-1 subtypes A-G, as well as HIV-2, simian immunodeficiency virus, and chimeric simian/human immunodeficiency virus (SHIV). Neutralization was demonstrated in both pre- and postinfection models. The administration of mAb B4 after infectious challenge totally interrupted the infection of hu-PBL-severe combined immunodeficient mice by PBL-grown HIV-1 and the infection of chimpanzees by chimp-adapted HIV-1. This mode of protection suggested that the anti-HIV receptor antibody is efficacious for prophylaxis after exposure to HIV and for prevention of maternal transmission and may be an effective antiretroviral agent for treatment.

Redefining the HIV-infectious window period in the chimpanzee model: evidence to suggest that viral nucleic acid testing can prevent blood-borne transmission.

BACKGROUND: Although it is rare, blood-transmitted HIV infection can occur when a donor presents in the window period between HIV-1 exposure and the first appearance of detectable p24 antigen. STUDY DESIGN AND METHODS: To study this seronegative window period, a chimpanzee (X034) was inoculated with 38 median tissue culture infective doses of HIV-1 IIIB; serum and peripheral blood mononuclear cells were obtained one to two times per week for 12 weeks and then biweekly for 12 weeks. Infectivity was monitored by the detection of serum HIV RNA, cell-associated HIV DNA, p24 antigen, and anti-HIV and by co-culture methods. RESULTS: No HIV markers were noted until 5 weeks after inoculation, at which time virus was isolated and HIV RNA and DNA were detected in plasma and cells, respectively. Anti-HIV and HIV p24 antigen were not present until 8 weeks after inoculation. Plasma and cells obtained from Chimpanzee X034 3 or 4 weeks after exposure were then sequentially inoculated into a second chimpanzee (X176); no HIV infection was observed in this animal during serial follow-up for 24 weeks after each inoculation. In contrast, when the fifth-week HIV-1 RNA- and DNA-positive sample was inoculated, Chimpanzee X176 was unequivocally infected with HIV-1. CONCLUSIONS: Nucleic acid testing narrowed the seronegative window by 3 weeks (37%). More important, there was no demonstrable infectivity in either plasma or peripheral blood mononuclear cells obtained before molecular markers were detectable. This suggests that the infectious window may be considerably shorter than the total window as measured from exposure and that nucleic acid testing might not only shorten the seronegative window, but totally prevent transfusion-transmitted HIV infection.

ZRP-1, a zyxin-related protein, interacts with the second PDZ domain of the cytosolic protein tyrosine phosphatase hPTP1E.

Protein-protein interactions play an important role in the specificity of cellular signaling cascades. By using the yeast two-hybrid system, a specific interaction was identified between the second PDZ domain of the cytosolic protein tyrosine phosphatase hPTP1E and a novel protein, which was termed ZRP-1 to indicate its sequence similarity to the Zyxin protein family. The mRNA encoding this protein is distributed widely in human tissues and contains an open reading frame of 1428 base pairs, predicting a polypeptide of 476 amino acid residues. The deduced protein displays a proline-rich amino-terminal region and three double zinc finger LIM domains at its carboxyl terminus. The specific interaction of this novel protein with the second PDZ domain of hPTP1E was demonstrated both in vitro, using bacterially expressed proteins, and in vivo, by co-immunoprecipitation studies. Deletion analysis indicated that an intact carboxyl terminus is required for its interaction with the second PDZ domain of hPTP1E in the yeast two-hybrid system and suggested that other sequences, including the LIM domains, also participate in the interaction. The genomic organization of the ZRP-1 coding sequence is identical to that of the lipoma preferred partner gene, another Zyxin-related protein, suggesting that the two genes have evolved from a recent gene duplication event.

The resistance of retroviral vectors produced from human cells to serum inactivation in vivo and in vitro is primate species dependent.

The ability to deliver genes as therapeutics requires an understanding of the vector pharmacokinetics similar to that required for conventional drugs. A first question is the half-life of the vector in the bloodstream. Retroviral vectors produced in certain human cell lines differ from vectors produced in nonhuman cell lines in being substantially resistant to inactivation in vitro by human serum complement (F. L. Cosset, Y. Takeuchi, J. L. Battini, R. A. Weiss, and M. K. Collins, J. Virol. 69:7430-7436, 1995). Thus, use of human packaging cell lines (PCL) may produce vectors with longer half-lives, resulting in more-efficacious in vivo gene therapy. However, survival of human PCL-produced vectors in vivo following systemic administration has not been explored. In this investigation, the half-lives of retroviral vectors packaged by either canine D17 or human HT1080 PCL were measured in the bloodstreams of macaques and chimpanzees. Human PCL-produced vectors exhibited significantly higher concentrations of circulating biologically active vector at the earliest time points measured (>1, 000-fold in chimpanzees), as well as substantially extended half-lives, compared to canine PCL-produced vectors. In addition, the circulation half-life of human PCL-produced vector was longer in chimpanzees than in macaques. This was consistent with in vitro findings which demonstrated that primate serum inactivation of vector produced from human PCL increased with increasing phylogenetic distance from humans. These results establish that in vivo retroviral vector half-life correlates with in vitro resistance to complement. Furthermore, these findings should influence the choice of animal models used to evaluate retroviral-vector-based therapies.

Fusion proteins could generate false positives in peptide phage display.

Fusion proteins are frequently used in the functional characterization of newly discovered proteins and to identify interacting partners. In our study of hPTP1E, a cytosolic protein tyrosine phosphatase, we used glutathione S-transferase (GST)-fusion protein of the second PDZ domain to identify interacting peptide motifs by peptide phage display. A consensus motif G X X V W L G was identified and found to be specific for binding to GST-PDZ2 as determined by ELISA, peptide displacement and by protein overlay. However, using nuclear magnetic resonance (NMR), no interaction of the peptide was observed with PDZ2 alone. In co-precipitation experiments using the consensus peptide cross-linked to Affi-Gel, only GST-PDZ2 (but not PDZ2 or GST alone) could be precipitated. These data suggest that there is a potential for identification of artifacts when using fusion proteins in peptide phage display, and one should exercise caution in interpreting these results. It is critical that the interaction be verified using a second, independent system.

Active and passive immunization against HIV type 1 infection in chimpanzees.

Several vaccine strategies have been developed to prevent primary infection with human immunodeficiency virus (HIV), and some of the candidate vaccines have been tested in chimpanzees to determine their safety, efficacy, and to delineate immune correlates of protection. To date, more than 25 vaccines representing active and passive immunization strategies have been evaluated in the chimpanzee model. Efficacy of a given vaccine was based on protection against primary infection with HIV after intravenous or mucosal challenge with cell-free or cell-associated virus. Based on the results from a majority of the studies, neutralizing antibodies appear to play a major role in preventing primary infection with HIV.

Genetic immunization of chimpanzees chronically infected with the hepatitis B virus, using a recombinant retroviral vector encoding the hepatitis B virus core antigen.

Cytotoxic T lymphocyte (CTL) activity and CD4+ helper T cell responses to the hepatitis B virus (HBV) core antigen (HBcAg) have been implicated in clearance of acute and chronic HBV infections. We showed that intramuscular injections of a novel recombinant retroviral vector expressing an HBcAg-neomycin phosphotransferase II (HBc-NEO) fusion protein induces HBc/eAg-specific antibodies and CD4+ and CD8+ T cell responses in mice and rhesus monkeys. We have now immunized three chronically infected chimpanzees, each with 10(10) CFU of nonreplicating retroviral vector particles expressing the HBc-NEO fusion protein. Of two immunized chimpanzees examined for CTL responses, one developed HBcAg-specific CTLs and showed marginal, transient elevations of alanine aminotransferase (ALT) levels following injection. However, both chimpanzees remained positive for serum HBeAg, negative for anti-HBe antibody by conventional assays, and displayed no change in HBV viral load throughout the study. In contrast, the third chimpanzee exhibited a traditional seroconversion evidenced by a loss of serum HBeAg and the subsequent emergence of anti-HBe antibodies within 24 weeks after the first injection. Simultaneously, two transient ALT flares and a significant decrease in the serum HBV DNA levels were noted. Despite its limitations, the present study demonstrates (1) the safety of treatment with high titers of retroviral vector in chimpanzees, (2) the capability of a retroviral vector expressing HBcAg to stimulate immune responses in HBV chronic carrier chimpanzees, and (3) that retroviral vector immunization may be therapeutically beneficial in the treatment of chronic HBV infection.

Epitope mapping of SHP-1 monoclonal antibodies using peptide phage display.

We have characterized the binding epitopes of four monoclonal antibodies for SHP-1, an SH2 domain containing protein tyrosine phosphatase, using two phage displayed random peptide libraries. Three of the antibodies are directed against the phosphatase domain of the molecule and the fourth is toward the NH2-terminal part of the second SH2 domain. The first two antibodies recognize the sequence NANY, amino acid 305 to amino acid 308, numbered in the non haematopoietic form of human SHP-1 sequence. The third antibody binds the sequence P Y W P (amino acids 365 to 368) located toward the middle of the phosphatase domain of the enzyme. The fourth antibody is directed against the first two amino acids, W Y (amino acids 112 and 113), of the second SH2 domain. The specificities of these antibodies are demonstrated by ELISA and western blot using different protein constructs expressed in bacteria. All the antibodies can detect wild type SHP-1, expressed in 293 cells, by western blot analysis, both under denaturing conditions as well as following renaturation. The data presented here show that the antibodies characterized in this study are raised against linear epitopes and suggest that these epitopes are accessible from the outside in the native SHP-1 molecule.

Human immunodeficiency virus-2 infection in baboons is an animal model for human immunodeficiency virus pathogenesis in humans.

OBJECTIVE: To assess disease progression in baboons (Papio cynocephalus) that were infected with two human immunodeficiency virus-2 (HIV-2) isolates. METHODS: Eight baboons were inoculated intravenously with either HIV-2UC2 or HIV-2UC14 and were followed for a 2- to 7-year period of observation. RESULTS: Six of 8 baboons showed lymphadenopathy and other signs of HIV-related disease, 3 of 8 baboons had an acute phase CD4+ T-cell decline, and 2 of 5 baboons infected with the HIV-2UC2 isolate progressed to an acquired immunodeficiency syndrome-like disease. Human immunodeficiency virus-2-specific pathology in lymphatic tissues included follicular lysis, vascular proliferation, and lymphoid depletion. Both neutralizing antibodies and a CD8+ T-cell antiviral response were associated with resistance to disease. CONCLUSIONS: Disease progression and the development of acquired immunodeficiency syndrome in HIV-2-infected baboons have similarities to human HIV infections.

Transient virus infection and pathogenesis of a new HIV type 2 isolate, UC12, in baboons.

We have previously shown that baboons (Papio cynocephalus) can be persistently infected with HIV-2 and some baboons progress to an AIDS-like disease with a CD4+ T cell decline, cachexia, alopecia, and Kaposi's sarcoma-like fibromatosis. In this study, we found that a new virus isolate, HIV-2UC12, replicated to high levels in baboon peripheral blood mononuclear cells (PBMCs) in vitro. Three baboons were subsequently inoculated and had plasma viral RNA loads that peaked between 15,000 and 7000 copies/ml at 2 weeks postinfection. Virus was isolated from the PBMCs for up to 6 months. Although PBMCs were subsequently virus culture negative, virus could be recovered from the spleen, lymph nodes, and tonsils, indicating that HIV-2 was sequestered within these lymphoid tissues. HIV-2-associated pathology included follicular lysis, vascular proliferation, and lymphoid depletion. This study indicated that HIV-2UC12 infection in baboons can cause HIV-associated pathological abnormalities within the lymphatic tissues and that the high level of HIV-2UC12 replication in vitro was not predictive of replication in vivo.

A synthetic peptide from the first conserved region in the envelope protein gp160 is a strong T-cell epitope in HIV-infected chimpanzees and humans.

We reported earlier that synthetic peptides corresponding to highly conserved regions in the envelope protein gp160 of the human immunodeficiency virus type 1 (HIV-1), in particular an 11-amino acid sequence (peptide 104) from the first conserved region at the amino-terminus, were capable of inducing strong HIV-specific T-cell proliferative responses in several inbred mouse strains as well as in outbred Rhesus monkeys. We have now obtained evidence of the presence of significant levels of proliferative response to peptide 104 in 7 of 9 chimpanzees chronically infected with HIV-1 (p < or = 0.05) and 8 of 17 HIV+ individuals (p < or = 0.001). Further, four other conserved HIV envelope-derived peptides, identified previously in our murine and Rhesus monkey model systems, were widely recognized as T-cell epitopes in both chimpanzees and humans infected with HIV-1. In none of the infected subjects did peripheral blood mononuclear cells show proliferative responses to unrelated control peptides. Also, neither the control normal chimpanzees nor HIV-seronegative individuals showed proliferative responses to the conserved peptides. With respect to the humoral responses, serum samples from none of the chimpanzees showed reactivity with any of the conserved peptides, and only low levels of antibody responses against peptide 104 were observed in 3 of the 17 patients (p > 0.05). Importantly, three of the conserved envelope-derived peptides, including peptide 104, overlap with sequences that were reported in the literature to be epitopes for virus-induced cytotoxic T lymphocytes in asymptomatic HIV+ individuals. These observations, together with our results in multiple animal models and humans, establish that these conserved HIV envelope-derived peptides, particularly peptide 104, are significant T-cell epitopes with potential usefulness for induction of HIV-specific cell-mediated immune responses in humans.