Differential escape patterns within the dominant HLA-B*57:03-restricted HIV Gag epitope reflect distinct clade-specific functional constraints.

UNLABELLED: HLA-B*57:01 and HLA-B*57:03, the most prevalent HLA-B*57 subtypes in Caucasian and African populations, respectively, are the HLA alleles most protective against HIV disease progression. Understanding the mechanisms underlying this immune control is of critical importance, yet they remain unclear. Unexplained differences are observed in the impact of the dominant cytotoxic T lymphocyte (CTL) response restricted by HLA-B*57:01 and HLA-B*57:03 in chronic infection on the Gag epitope KAFSPEVIPMF (KF11; Gag 162 to 172). We previously showed that the HLA-B*57:03-KF11 response is associated with a >1-log-lower viral setpoint in C clade virus infection and that this response selects escape mutants within the epitope. We first examined the relationship of KF11 responses in B clade virus-infected subjects with HLA-B*57:01 to immune control and observed that a detectable KF11 response was associated with a >1-log-higher viral load (P = 0.02). No evidence of HLA-B*57:01-KF11-associated selection pressure was identified in previous comprehensive analyses of >1,800 B clade virus-infected subjects. We then studied a B clade virus-infected cohort in Barbados, where HLA-B*57:03 is highly prevalent. In contrast to findings for B clade virus-infected subjects expressing HLA-B*57:01, we observed strong selection pressure driven by the HLA-B*57:03-KF11 response for the escape mutation S173T. This mutation reduces recognition of virus-infected cells by HLA-B*57:03-KF11 CTLs and is associated with a >1-log increase in viral load in HLA-B*57:03-positive subjects (P = 0.009). We demonstrate functional constraints imposed by HIV clade relating to the residue at Gag 173 that explain the differential clade-specific escape patterns in HLA-B*57:03 subjects. Further studies are needed to evaluate the role of the KF11 response in HLA-B*57:01-associated HIV disease protection. IMPORTANCE: HLA-B*57 is the HLA class I molecule that affords the greatest protection against disease progression in HIV infection. Understanding the key mechanism(s) underlying immunosuppression of HIV is of importance in guiding therapeutic and vaccine-related approaches to improve the levels of HIV control occurring in nature. Numerous mechanisms have been proposed to explain the HLA associations with differential HIV disease outcome, but no consensus exists. These studies focus on two subtypes of HLA-B*57 prevalent in Caucasian and African populations, HLA-B*57:01 and HLA-B*57:03, respectively. These alleles appear equally protective against HIV disease progression. The CTL epitopes presented are in many cases identical, and the dominant response in chronic infection in each case is to the Gag epitope KF11. However, there the similarity ends. This study sought to better understand the reasons for these differences and what they teach us about which immune responses contribute to immune control of HIV infection.

Widespread expression of the human and rat Huntington's disease gene in brain and nonneural tissues.

We have used RNA in situ hybridization to study the regional expression of the Huntington's disease gene (HD) and its rat homologue in brain and selected nonneural tissues. The HD transcript was expressed throughout the brain in both rat and human, especially in the neurons of the dentate gyrus and pyramidal neurons of the hippocampal formation, cerebellar granule cell layer, cerebellar Purkinje cells and pontine nuclei. Other brain areas expressed lower levels of the HD transcript without pronounced regional differences. Neuronal expression predominated over glial expression in all regions. HD mRNA was also expressed in colon, liver, pancreas and testes. The regional specificity of neuropathology in HD, which is most prominent in the basal ganglia, thus cannot be accounted for by the pattern of expression of HD.

Chloride conductance expressed by delta F508 and other mutant CFTRs in Xenopus oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR) is associated with expression of a chloride conductance that is defective in cystic fibrosis (CF). Xenopus oocytes injected with RNA coding for CFTR that contained mutations in the first nucleotide binding fold (NBF1) expressed chloride currents in response to raising adenosine 3',5'-monophosphate (cAMP) with forskolin and 3-isobutyl-1-methylxanthine (IBMX). The mutant CFTRs were less sensitive than wild-type CFTR to this activating stimulus, and the reduction in sensitivity correlated with the severity of cystic fibrosis in patients carrying the corresponding mutations. This demonstration provides the basis for detailed analyses of NBF1 function and suggests potential pharmacologic treatments for cystic fibrosis.

Lack of effect of a topical regenerative agent on re-epithelialization rate of canine spontaneous chronic corneal epithelial defects: A randomized, double-masked, placebo-controlled study.

Spontaneous chronic corneal epithelial defects (SCCEDs) are characteristic ulcers in dogs that are refractory to healing. The aim of the study was to evaluate the use of a topical regenerative agent to promote healing of SCCEDs. Nineteen dogs (20 eyes) were randomized to receive either regenerative agent (10 eyes) or placebo (10 eyes) every 48h following corneal debridement, which was repeated 1 week later if the SCCED had not yet healed. The mean±standard deviation time to re-epithelialization was 17.3±12.8 days for the group treated with a topical regenerative agent and 19.3±11.7 days for the group treated with a placebo; the cumulative healing rates were not statistically different (P>0.650). A positive association was found between the initial size of the ulcer and the time to re-epithelialization (r=0.555, P=0.011). Although well tolerated by dogs, there was no therapeutic advantage in using a topical regenerative agent for re-epithelialization of SCCEDs.

Localization of cystic fibrosis transmembrane conductance regulator mRNA in the human gastrointestinal tract by in situ hybridization.

We have used in situ hybridization to localize expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in the human gastrointestinal tract and associated organs. The stomach exhibits a low level of CFTR expression throughout gastric mucosa. In the small intestine, expression is relatively high in the mucosal epithelium, with a decreasing gradient of expression along the crypt to tip axis. The cells of the Brunner's glands express high levels of CFTR mRNA. In addition, there is a small subpopulation of highly positive cells scattered along the epithelium in the duodenum and jejunum, but not in the ileum. These cells do not represent endocrine cells, as determined by lack of colocalization with an endocrine-specific marker. The distribution of CFTR mRNA in the colon is similar to the small intestine, with highest level of expression in the epithelial cells at the base of the crypts. In the pancreas, CFTR is expressed at high levels in the small, intercalated ducts and at lower levels in the interlobular ducts. CFTR transcripts are expressed at uniformly high levels in the epithelium of the gallbladder. Throughout the gastrointestinal tract, CFTR expression is increased in mucosal epithelial cells that are near lymph nodules.

Evidence for tau expression in cells of monocyte lineage and its in vitro phosphorylation by v-fms kinase.

The v-fms encoded kinase, which is known to associate with cytoskeletal elements, was shown to phosphorylate histiocytic proteins that had physical and immunological attributes in common with tau. Tau is a microtubule associated protein that is aberrantly phosphorylated in Alzheimer's disease of the brain. Since c-fms expression is normally exclusive to cells of monocyte lineage, these cells were examined for expression of tau. Heat stable tau was identified in cultured peripheral blood monocytes. This represents the first molecular characterization of tau in cells of non-neural origin.

Expression of human endogenous retrovirus k envelope transcripts in human breast cancer.

PURPOSE: We investigated the expression of human endogenous retroviral (HERV) sequences in breast cancer. EXPERIMENTAL DESIGN: Reverse transcription-PCR (RT-PCR) was used to examine expression of the envelope (env) region of ERV3, HERV-E4-1, and HERV-K in breast cancer cell lines, human breast tumor samples, adjacent uninvolved breast tissues, nonmalignant breast tissues, and placenta. Expression of HERV transcripts was confirmed by Northern blot analysis and in situ hybridization (ISH). To evaluate coding potential, amplified HERV sequences were cloned into vectors for expression and sequence analysis. RESULTS: No expression of ERV3 or HERV-E4-1 RNA was detected in the analyzed breast samples. In contrast, HERV-K transcripts were detected in most breast cancer cell lines and many breast tumor tissues. Expression was detected in a small percentage of matched, uninvolved breast tissues and in placentas but not nonmalignant breast tissues. In HERV-K-positive breast cancer tissues, Northern blot analysis demonstrated full-length proviral and spliced env transcripts. ISH demonstrated expression of HERV-K transcripts in breast tumor cells but not in normal or uninvolved breast epithelial cells. Independently isolated clones of HERV-K env cDNA generated recombinant proteins of the expected size. Sequence analysis of env cDNA clones derived from four breast tumor samples revealed >97% identity with the type I HERV-K102, with no premature termination codons. Independent isolates from the same breast tumor sample showed nucleotide sequence differences, suggesting that multiple loci may be transcribed. CONCLUSIONS: These data indicate that HERV-K transcripts with coding potential for the envelope region are expressed frequently in human breast cancer.

A cancer gene therapy approach utilizing an anti-erbB-2 single-chain antibody-encoding adenovirus (AD21): a phase I trial.

The purpose of this Phase I study was to determine the feasibility of using an anti-erbB-2-encoding adenovirus (Ad21) to treat erbB-2-overexpressing ovarian cancer. Recurrent ovarian cancer patients were treated i.p. with Ad21 in dosages ranging from 1 x 10(9) to 1 x 10(11) pfu. Patients were monitored after treatment for evidence of clinical toxicity and efficacy. Peritoneal aspirates and serum samples were obtained to assess for evidence of gene transfer/expression, for generation of wild-type vector, and antiadenoviral humoral response. Fifteen patients were treated per study specifications. Treatment-specific grade 1/2 fever was experienced by 9 of 15 (60%) patients. Other transient grade 1/2 constitutional, pain, and gastrointestinal symptoms were also experienced. No dose-limiting vector-related toxicity was experienced. Of 13 patients evaluable for response, 5 (38%) had stable disease and 8 (61%) had evidence of progressive disease. One patient with nonmeasurable disease normalized her CA125 at the 8-week evaluation, and one patient with nonmeasurable disease remained without clinical evidence of disease for 6 months after treatment. PCR analysis of peritoneal aspirates demonstrated the presence of Ad21 in 84.6%, 84.6%, and 61.6% of evaluable specimens at days 2, 14, and 56 after treatment, respectively. No wild-type virus was detected. Reverse transcription-PCR analysis demonstrated expression of the anti-erbB-2 sFv-encoding gene in 10 of 14 evaluable patients at day 2. Five of six evaluable patients had an increase in antiadenovirus antibody titer. This study suggests that adenoviral-mediated gene therapy using an anti-erbB-2-directed intrabody is feasible in the context of human ovarian cancer.

Selective gene delivery to head and neck cancer cells via an integrin targeted adenoviral vector.

In vivo cancer gene therapy approaches for squamous cell carcinoma of the head and neck (SCCHN) based on adenoviral vector-mediated gene delivery have been limited by the suboptimal efficacy of gene transfer to tumor cells. We hypothesized that this issue was due to deficiency of the primary adenoviral receptor, the coxsackie-adenovirus receptor (CAR), on the tumor targets. Studies of CAR levels on SCCHN cell lines confirmed that their relative refractoriness to the adenoviral vector was based on this deficiency. To circumvent this deficiency, we applied an adenoviral vector targeted to a tumor cell marker characteristic of SCCHN. In this regard, integrins of the alpha2beta1 and alpha3beta1 class are frequently overexpressed in SCCHN. Furthermore, these integrins recognize the RGD peptide motif. On this basis, we applied an adenoviral vector genetically modified to contain such a peptide within the HI loop of the fiber protein as a means to alter viral tropism. Studies confirmed that the CAR-independent gene delivery achieved via this strategy allowed enhanced gene transfer efficiencies to SCCHN tumor cells. Importantly, this strategy could achieve preferential augmentation of gene transfer in tumor cells compared with normal cells. The ability to achieve enhanced and specific gene transfer to tumor cells via adenoviral vectors has important implications for gene therapy strategies for SCCHN and for other neoplasms in general.

Polynucleotide immunization of nonhuman primates against carcinoembryonic antigen.

In preparation for a Phase I trial of DNA immunization against carcinoembryonic antigen (CEA) in patients with colorectal carcinoma, we have produced a single plasmid DNA encoding CEA and hepatitis B surface antigen (HBsAg) under transcriptional regulatory control of two separate cytomegalovirus promoters within separate eukaryotic expression cassettes, designated pCEA/HBsAg. Hepatitis B surface antigen was included to provide an internal positive control for the efficacy of this immunization strategy without regard to the issue of breaking tolerance to a self-antigen. In the present work, we sought to examine the immunogenicity of this plasmid in a nonhuman primate model with close phylogenetic relationship to humans. Groups of pig-tailed macaques were immunized with pCEA/ HBsAg by i.m. injection or particle bombardment of the skin according to a dose and schedule thought to be optimal for the respective technique of DNA immunization. Both administration techniques produced humoral and lympho-proliferative responses of comparable magnitude. However, delayed type hypersensitivity to CEA and CEA-specific interleukin-2 release were observed only in the i.m. group, suggesting a qualitative difference in the character of the immune response elicited by the two techniques of DNA immunization. The antibody responses to CEA and HBsAg were surprisingly persistent in that all immunized animals maintained moderate antibody titers against both antigens for more than 15 months after the last boost. No toxicity was observed during 2 years of follow-up, including no measurable levels of anti-DNA antibody. This antitumor immunization strategy is presently being examined in patients with metastatic colorectal carcinoma using pCEA/HBsAg administered by i.m. injection.

Using a tropism-modified adenoviral vector to circumvent inhibitory factors in ascites fluid.

Peritoneal compartmentalization of advanced stage ovarian cancer provides a rational scenario for gene therapy strategies. Several groups are exploring intraperitoneal administration of adenoviral (Ad) vectors for this purpose. We examined in vitro gene transfer in the presence of ascites fluid from ovarian cancer patients and observed significant inhibition of Ad-mediated gene transfer. The inhibitory activity was not identified as either complement or cellular factors, but depletion of IgG from ascites removed the inhibitory activity, implicating neutralizing anti-Ad antibodies. A wide range of preexisting anti-Ad antibody titers in patient ascites fluid was measured by ELISA. Western blot analysis demonstrated that the antibodies were directed primarily against the Ad fiber protein. To circumvent inhibition by neutralizing antibodies, a genetically modified adenoviral vector was tested. The Ad5Luc.RGD vector has an Arg-Gly-Asp (RGD) peptide sequence inserted into the fiber knob domain and enters cells through a nonnative pathway. Compared with the conventional Ad5 vector, Ad5Luc.RGD directed efficient gene transfer to cell lines and primary ovarian cancer cells in the presence of ascites fluid containing high-titer neutralizing anti-Ad antibodies. These results suggest that such modified Ad vectors will be needed to achieve efficient gene transfer in the clinical setting.

SEREX analysis for tumor antigen identification in a mouse model of adenocarcinoma.

Evaluation of immunotherapy strategies in mouse models of carcinoma is hampered by the limited number of known murine tumor antigens (Ags). Although tumor Ags can be identified based on cytotoxic T-cell activation, this approach is not readily accomplished for many tumor types. We applied an alternative strategy based on a humoral immune response, SEREX, to the identification of tumor Ags in the murine colon adenocarcinoma cell line MC38. Immunization of syngeneic C57BL/6 mice with MC38 cells by three different methods induced a protective immune response with concomitant production of anti-MC38 antibodies. Immunoscreening of an MC38-derived expression library resulted in the identification of the endogenous ecotropic leukemia virus envelope (env) protein and the murine ATRX protein as candidate tumor Ags. Northern blot analysis demonstrated high levels of expression of the env transcript in MC38 cells and in several other murine tumor cell lines, whereas expression in normal colonic epithelium was absent. ATRX was found to be variably expressed in tumor cell lines and in normal tissue. Further analysis of the expressed env sequence indicated that it represents a nonmutated tumor Ag. Polynucleotide immunization with DNA encoding the env polypeptide resulted in strong and specific antibody responses to this self Ag in all immunized mice. Thus, SEREX offers a rapid means of identifying tumor Ags in murine cancer models.

The effect of amnioinfusion on the duration of labor.

OBJECTIVE: To test the hypothesis that women receiving intrapartum amnioinfusion have more rapid labors than do controls. DATA SOURCES: Prospective clinical trials of amnioinfusion published in major American obstetric and gynecologic journals between 1985 and 1995, identified through a literature search using MEDLINE and manual index review, were examined. METHOD OF STUDY SELECTION: Eleven studies that presented data regarding the length of labor were identified. Each study was reviewed for the design, number of subjects enrolled, volume of amnioinfusate, birth weight, maternal parity, interval from amniorrhexis to delivery, and total length of labor. TABULATION, INTEGRATION AND RESULTS: Meta-analysis revealed no differences between amnioinfusion groups and controls with regard to length of labor or the interval between membrane rupture and delivery. CONCLUSION: Amnioinfusion has no effect on the duration of labor.

The umbilical pump: a contributor to twin-twin transfusion.

BACKGROUND: Umbilical vascular coiling may function as a rudimentary pump that facilitates venous return from the placenta. CASES: Three consecutive twin gestations with twin-twin transfusion syndrome were evaluated prospectively at delivery. The birth weights and umbilical coiling indices of donor and recipient twins were compared. The umbilical coiling index was determined by dividing the number of complete vascular coils in a given umbilical cord by the cord's length in centimeters. In each case, the recipient twin was larger at birth and had an umbilical coiling index value that was at least twice that of the corresponding donor twin. CONCLUSION: Differential umbilical vascular coiling densities among monochorionic twins may play a role in the pathogenesis of twin-twin transfusion syndrome.

Intrapartum uterine activity: evaluation of an intrauterine pressure transducer.

A newly available intrauterine pressure transducer was evaluated clinically in 100 patients. Successful insertion was accomplished in 95%. There were no significant intrapartum maternal or fetal complications. Partial dehiscence of a surgically scarred uterus did occur in one patient who received the device, but a clear relationship between its attempted insertion and the dehiscence was not apparent. Early in the clinical trial, a number of devices malfunctioned; the manufacturer defined and remedied the problem. The intrauterine transducer required no maintenance and appeared to be practical in laboring women. We suggest that the utility of the intrauterine pressure transducer might be enhanced with several modifications, including the addition of a re-zeroing mechanism and a reduction in the device's length.

Medical and psychologic characteristics of women presenting with premenstrual symptoms.

To establish the necessary elements of a program of evaluation and treatment of premenstrual syndrome, the medical and psychologic characteristics of 68 consecutive women presenting because of premenstrual symptoms were determined and compared with those of a similar group of 34 women without premenstrual symptoms (control group). Women with premenstrual symptoms exhibited a significantly greater frequency of previously undetected medical, psychologic, and marital problems than did controls. These findings demonstrate the need for a multidisciplinary comprehensive program of evaluation and treatment of the medical, psychologic, and mental health of women who present because of moderate-to-severe premenstrual symptoms.

Non-coiled umbilical blood vessels: a new marker for the fetus at risk.

OBJECTIVE: To evaluate the perinatal outcomes of fetuses born with non-coiled umbilical blood vessels. METHODS: We performed a prospective study of umbilical cords that lacked umbilical vascular coiling. The perinatal outcomes were compared with those of neonates born with coiled umbilical blood vessels. RESULTS: Thirty-eight (4.3%) of 894 fetuses were born with non-coiled umbilical vessels. The non-coiled group had a significantly increased incidence of intrauterine death (P = .009), preterm delivery (P = .006), repetitive intrapartum fetal heart rate decelerations (P < .00005), operative delivery for fetal distress (P < .00005), meconium staining (P = .007), and anatomical-karyotypic abnormalities (P = .03). CONCLUSIONS: Our findings suggest that the fetus with non-coiled (ie, straight) umbilical blood vessels is at increased risk for perinatal morbidity and mortality. Non-coiled umbilical vessels may represent a pathologic developmental process that places the fetus at risk. Moreover, absence of the normal coiled umbilical configuration may result in a cord that is structurally less able to resist external compressive forces.

Amnioinfusion among women attempting vaginal birth after cesarean delivery.

Eighteen of 901 women (2%) attempting vaginal birth after cesarean delivery (VBAC) received amnioinfusion. No untoward effects occurred in the subjects or their fetuses. We conclude that, though amnioinfusion in the setting of a VBAC attempt is needed only infrequently, it appears to be a reasonable intrapartum management option. The usual safeguards for a VBAC attempt should be followed.

Fetal death from sepsis following a reassuring intrapartum fetal acoustic stimulation test.

An intrapartum fetal death within 20 minutes of a reassuring acoustically stimulated fetal heart rate acceleration is reported. The cause of death in this instance was congenital pneumonia, gram-negative sepsis, and meconium aspiration. Umbilical cord pH values obtained at delivery did not demonstrate asphyxia (ie, low pO2, high pCO2, and low pH), but suggested a metabolic acidosis typical of sepsis.

Congenital depression of the fetal skull.

One hundred forty-seven cases of congenital skull depression are analyzed, including two presented by the authors, and a review of the literature follows. A management plan emphasizing a conservative approach is outlined.