Codon optimization and improved delivery/immunization regimen enhance the immune response against wild-type and drug-resistant HIV-1 reverse transcriptase, preserving its Th2-polarity.

DNA vaccines require a considerable enhancement of immunogenicity. Here, we optimized a prototype DNA vaccine against drug-resistant HIV-1 based on a weak Th2-immunogen, HIV-1 reverse transcriptase (RT). We designed expression-optimized genes encoding inactivated wild-type and drug-resistant RTs (RT-DNAs) and introduced them into mice by intradermal injections followed by electroporation. RT-DNAs were administered as single or double primes with or without cyclic-di-GMP, or as a prime followed by boost with RT-DNA mixed with a luciferase-encoding plasmid ("surrogate challenge"). Repeated primes improved cellular responses and broadened epitope specificity. Addition of cyclic-di-GMP induced a transient increase in IFN-γ production. The strongest anti-RT immune response was achieved in a prime-boost protocol with electroporation by short 100V pulses done using penetrating electrodes. The RT-specific response, dominated by CD4+ T-cells, targeted epitopes at aa 199-220 and aa 528-543. Drug-resistance mutations disrupted the epitope at aa 205-220, while the CTL epitope at aa 202-210 was not affected. Overall, multiparametric optimization of RT strengthened its Th2- performance. A rapid loss of RT/luciferase-expressing cells in the surrogate challenge experiment revealed a lytic potential of anti-RT response. Such lytic CD4+ response would be beneficial for an HIV vaccine due to its comparative insensitivity to immune escape.

Assessment of the Parameters of Adaptive Cell-Mediated Immunity in Naïve Common Marmosets (Callithrix jacchus).

Common marmosets are small New World primates that have been increasingly used in biomedical research. This report presents efficient protocols for assessment of the parameters of adaptive cell-mediated immunity in common marmosets, including the major subpopulations of lymphocytes and main markers of T- and B-cell maturation and activation using flow cytometry with a multicolor panel of fluorescently labelled antibodies. Blood samples from eight common marmosets were stained with fluorescently labeled monoclonal antibodies against their population markers (CD45, CD3, CD20, CD4, CD8) and lymphocyte maturation and activation markers (CD69, CD62L, CD45RO, CD107a and CD27) and analyzed by flow cytometry. Within the CD45+ population, 22.7±5.5% cells were CD3- CD20+ and 67.6±6.3% were CD3+CD20-. The CD3+ subpopulation included 55.7±5.5% CD3+CD4+CD8- and 34.3±3.7% CD3+CD4-CD8+ cells. Activation and maturation markers were expressed in the following lymphocyte proportions: CD62L on 54.0±10.7% of CD3+CD4+ cells and 74.4±12.1% of CD3+CD8+ cells; CD69 on 2.7±1.2% of CD3+CD4+ cells and 1.2±0.5% of CD3+CD8+ cells; CD45RO on 1.6±0.6% of CD3+CD4+ cells and 1.8±0.7% of CD3+CD8+ cells; CD107a on 0.7±0.5% of CD3+CD4+ cells and 0.5±0.3% of CD3+CD8+ cells; CD27 on 94.6±2.1% of CD3+ cells and 8.9±3.9% CD20+ cells. Female and male subjects differed in the percentage of CD3+CD4+CD45RO+ cells (1.9±0.5 in females vs 1.1±0.2 in males; p < 0.05). The percentage of CD20+CD27+ cells was found to highly correlate with animals' age (r = 0.923, p < 0.005). The basal parameters of adaptive cell-mediated immunity in naïve healthy marmosets without markers of systemic immune activation were obtained. These parameters and the described procedures are crucial in documenting the changes induced in common marmosets by prophylactic and therapeutic immune interventions.

Luciferase Expression Allows Bioluminescence Imaging But Imposes Limitations on the Orthotopic Mouse (4T1) Model of Breast Cancer.

Implantation of reporter-labeled tumor cells in an immunocompetent host involves a risk of their immune elimination. We have studied this effect in a mouse model of breast cancer after the orthotopic implantation of mammary gland adenocarcinoma 4T1 cells genetically labelled with luciferase (Luc). Mice were implanted with 4T1 cells and two derivative Luc-expressing clones 4T1luc2 and 4T1luc2D6 exhibiting equal in vitro growth rates. In vivo, the daughter 4T1luc2 clone exhibited nearly the same, and 4T1luc2D6, a lower growth rate than the parental cells. The metastatic potential of 4T1 variants was assessed by magnetic resonance, bioluminescent imaging, micro-computed tomography, and densitometry which detected 100-µm metastases in multiple organs and bones at the early stage of their development. After 3-4 weeks, 4T1 generated 11.4 ± 2.1, 4T1luc2D6, 4.5 ± 0.6; and 4T1luc2, <1 metastases per mouse, locations restricted to lungs and regional lymph nodes. Mice bearing Luc-expressing tumors developed IFN-γ response to the dominant CTL epitope of Luc. Induced by intradermal DNA-immunization, such response protected mice from the establishment of 4T1luc2-tumors. Our data show that natural or induced cellular response against the reporter restricts growth and metastatic activity of the reporter-labelled tumor cells. Such cells represent a powerful instrument for improving immunization technique for cancer vaccine applications.

Immunization with HIV-1 envelope T20-encoding DNA vaccines elicits cross-clade neutralizing antibody responses.

BACKGROUND: Genetic immunization is expected to induce the expression of antigens in a native form. The encoded peptide epitopes are presented on endogenous MHC molecules, mimicking antigen presentation during a viral infection. We have explored the potential of enfuvirtide (T20), a short HIV peptide with antiviral properties, to enhance immune response to HIV antigens. To generate an expression vector, the T20 sequence was cloned into a conventional plasmid, the novel minicircle construct, and a replicon plasmid. In addition, 3 conventional plasmids that express the envelope of HIV-1 subtypes A, B and C and contain T20 in their gp41 sequences were also tested. RESULTS: All combinations induced HIV-specific antibodies and cellular responses. The addition of T20 as a peptide and as an expression cassette in the 3 DNA vectors enhanced antibody responses. The highest anti-HIV-1 Env titers were obtained by the replicon T20 construct. This demonstrates that besides its known antiviral activity, T20 promotes immune responses. We also confirm that the combination of slightly divergent antigens improves immune responses. CONCLUSIONS: The antiretroviral T20 HIV-1 sequence can be used as an immunogen to elicit binding and neutralizing antibodies against HIV-1. These, or similarly modified gp41 genes/peptides, can be used as priming or boosting components for induction of broadly neutralizing anti-HIV antibodies. Future comparative studies will reveal the optimal mode of T20 administration.

HIVIS-DNA or HIVISopt-DNA priming followed by CMDR vaccinia-based boosts induce both humoral and cellular murine immune responses to HIV.

BACKGROUND: In order to develop a more effective prophylactic HIV-1 vaccine it is important optimize the components, improve Envelope glycoprotein immunogenicity as well as to explore prime-boost immunization schedules. It is also valuable to include several HIV-1 subtype antigens representing the world-wide epidemic. METHODS: HIVIS-DNA plasmids which include Env genes of subtypes A, B and C together with Gag subtypes A and B and RTmut/Rev of subtype B were modified as follows: the Envelope sequences were shortened, codon optimized, provided with an FT4 sequence and an immunodominant region mutated. The reverse transcriptase (RT) gene was shortened to contain the most immunogenic N-terminal fragment and fused with an inactivated viral protease vPR gene. HIVISopt-DNA thus contains fewer plasmids but additional PR epitopes compared to the native HIVIS-DNA. DNA components were delivered intradermally to young Balb/c mice once, using a needle-free Biojector® immediately followed by dermal electroporation. Vaccinia-based MVA-CMDR boosts including Env gene E and Gag-RT genes A were delivered intramuscularly by needle, once or twice. RESULTS: Both HIVIS-DNA and HIVISopt-DNA primed humoral and cell mediated responses well. When boosted with heterologous MVA-CMDR (subtypes A and E) virus inhibitory neutralizing antibodies were obtained to HIV-1 subtypes A, B, C and AE. Both plasmid compositions boosted with MVA-CMDR generated HIV-1 specific cellular responses directed against HIV-1 Env, Gag and Pol, as measured by IFNγ ELISpot. It was shown that DNA priming augmented the vector MVA immunological boosting effects, the HIVISopt-DNA with a trend to improved (Env) neutralization, the HIVIS-DNA with a trend to better (Gag) cell mediated immune reponses. CONCLUSIONS: HIVIS-DNA was modified to obtain HIVISopt-DNA that had fewer plasmids, and additional epitopes. Even with one DNA prime followed by two MVA-CMDR boosts, humoral and cell-mediated immune responses were readily induced by priming with either DNA construct composition. Priming by HIV-DNA augmented neutralizing antibody responses revealed by boosting with the vaccinia-based heterologous sequences. Cellular and antibody responses covered selected strains representing HIV-1 subtypes A, B, C and CRF01_AE. We assume this is related to the inclusion of heterologous full genes in the vaccine schedule.

Novel Clostridium perfringens enterotoxin suicide gene therapy for selective treatment of claudin-3- and -4-overexpressing tumors.

Bacterial toxins are known to be effective for cancer therapy. Clostridium perfringens enterotoxin (CPE) is produced by the bacterial Clostridium type A strain. The transmembrane proteins claudin-3 and -4, often overexpressed in numerous human epithelial tumors (for example, colon, breast, pancreas, prostate and ovarian), are the targeted receptors for CPE. CPE binding to them triggers formation of membrane pore complexes leading to rapid cell death. In this study, we aimed at selective tumor cell killing by CPE gene transfer. We generated expression vectors bearing the bacterial wild-type CPE cDNA (wtCPE) or translation-optimized CPE (optCPE) cDNA for in vitro and in vivo gene therapy of claudin-3- and -4-overexpressing tumors. The CPE expression analysis at messenger RNA and protein level revealed more efficient expression of optCPE compared with wtCPE. Expression of optCPE showed rapid cytotoxic activity, hightened by CPE release as bystander effect. Cytotoxicity of up to 100% was observed 72 h after gene transfer and is restricted to claudin-3-and -4-expressing tumor lines. MCF-7 and HCT116 cells with high claudin-4 expression showed dramatic sensitivity toward CPE toxicity. The claudin-negative melanoma line SKMel-5, however, was insensitive toward CPE gene transfer. The non-viral intratumoral in vivo gene transfer of optCPE led to reduced tumor growth in MCF-7 and HCT116 tumor-bearing mice compared with the vector-transfected control groups. This novel approach demonstrates that CPE gene transfer can be employed for a targeted suicide gene therapy of claudin-3- and -4-overexpressing tumors, leading to the rapid and efficient tumor cell killing in vitro and in vivo.

Unrecognised dural puncture resulting in subdural hygroma and cortical vein thrombosis.

Unrecognised dural punctures are difficult to diagnose early. Failure of recognition may lead to sinister consequences. A case of unrecognised dural puncture in a young female leading to the development of subdural hygroma and cortical vein thrombosis is presented. The dilemma in the diagnosis of headache in such patients along with the significance of follow-up of all, including attempted, epidurals is also discussed.

From zygote to implantation: morphological and molecular dynamics during embryo development in the pig.

The increasing focus on the pig as a biomedical model calls for studies which investigate morphological and molecular mechanisms during initial embryonic development in this species. In the pig, the paternal genome is actively demethylated in the zygote, whereas the maternal genome remains methylated. The major genome activation occurs at the four-cell stage, when prominent ribosome-synthesizing nucleoli develop in the blastomeres, allowing for trophectoderm and inner cell mass (ICM) differentiation. Unlike in mice, the pluripotency gene OCT4 is initially expressed in both compartments. The ICM differentiates into epiblast and hypoblast approximately at the time of hatching from the zona pellucida, and subsequently the loss of the Rauber's layer results in an uncovered epiblast establishing the embryonic disc again in contrast to mice. This particular and protracted ICM/epiblast biology may contribute to the lack of success in culturing porcine embryonic stem cells. The embryonic disc subsequently becomes polarized by a posterior thickening, which includes ingression of the first extra-embryonic mesoderm. Thereafter, the primitive streak forms and gastrulation results in formation of the somatic germ layers and germline, i.e. the primordial germ cells. The latter remain pluripotent for a period and may be isolated and cultured as embryonic germ cells in vitro.

Quantitative assessment of uncertainty in the optimization of metabolic pathways.

The S-System formalism provides a popular, versatile and mathematically tractable representation of metabolic pathways. At steady-state, after a logarithmic transformation, the S-System representation reduces into a system of linear equations. Thus, the maximization of a particular metabolite concentration or a flux subject to physiological constraints can be expressed as a linear programming (LP) problem which can be solved explicitly and exactly for the optimum enzyme activities. So far, the quantitative effect of parametric/experimental uncertainty on the S-model predictions has been largely ignored. In this work, for the first time, the systematic quantitative description of modeling/experimental uncertainty is attempted by utilizing probability density distributions to model the uncertainty in assigning a unique value to system parameters. This probabilistic description of uncertainty renders both objective and physiological constraints stochastic, demanding a probabilistic description for the optimization of metabolic pathways. Based on notions from chance-constrained programming and statistics, a novel approach is introduced for transforming the original stochastic formulation into a deterministic one which can be solved with existing optimization algorithms. The proposed framework is applied to two metabolic pathways characterized with experimental and modeling uncertainty in the kinetic orders. The computational results indicate the tractability of the method and the significant role that modeling and experimental uncertainty may play in the optimization of networks of metabolic reactions. While optimization results ignoring uncertainty sometimes violate physiological constraints and may fail to correctly assess objective targets, the proposed framework provides quantitative answers to questions regarding how likely it is to achieve a particular metabolic objective without exceeding a prespecified probability of violating the physiological constraints. Trade-off curves between metabolic objectives, probabilities of meeting these objectives, and chances of satisfying the physiological constraints, provide a concise and systematic way to guide enzyme activity alterations to meet an objective in the face of modeling and experimental uncertainty. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 145-161, 1997.

The trauma-surgery interval--implications for the outcome of traumatic intracranial hematomas.

The time that elapses between trauma and the beginning of surgical treatment is the interval during which traumatic intracranial hematomas are clinically manifested and diagnosed and the patients are prepared for surgical treatment. This trauma-surgery interval is divided into two periods: a diagnostic period and a period of preparation for surgery. A total of 610 patients treated in the Neurosurgery Department of the Plovdiv University of Medicine between 1975 and 1990 were retrospectively studied. They are allocated into groups of patients who survived or died during the trauma-surgery interval and patients treated during the consecutive stages of diagnostic capabilities of the Department: 1975-1979, 1980-1981, and 1982-1990. The study suggests that utilization of all methods of making a diagnose results in early identification of the hematoma and shortening of the diagnostic period of the trauma-surgery interval. This enables surgeons to operate on patients with acute hematomas at the earliest possible time after the trauma, achieving better therapeutic results. In patients with chronic hematomas the shorter diagnostic period makes it possible to devote the relatively expanded part of the trauma-surgery interval for preoperative preparation during which some concomitant disorders can be treated. Thus the risks from general anesthesia and the operative interventions are reduced.

A case of interhemispheric subdural hematoma.

The interhemispheric subdural hematoma is a rare condition. We present a case of interhemispheric subdural hematoma in a patient aged 65 years. A day prior to admission he was struck with a water-pipe on the head. He went to sleep the same evening complaining of a slight headache. At about two o'clock in the morning the headache increased in intensity. By the morning he lost consciousness. On examination by a neurosurgeon the patient was found to be comatose. The physical examination revealed blue eyelids of the left eye, paraplegia of the right leg, paresis of the left leg and arms. Bilateral Babinski's reflex was present, the abdominal reflexes were absent, the tendon and periosteal reflexes were hyperactive. The pupils were equal in size and slowly reactive to light. The patient exhibited symptoms of meningoradicular irritation. An emergency CT scan revealed high-density area in the interhemispheric sulcus extending frontally to parietally. The patients was operated on in an emergency. At operation, extensive rupture of the sagittal sinus was identified. Later the patient died. The presented case was interesting with the extensive rupture of the sagittal sinus and the relatively long lucid interval until clear manifestation of the clinical picture becomes evident.

Diagnosis and treatment of traumatic intracranial hematomas and hydromas according to the hospital type.

The capability to diagnose and treat is essentially a combination of the professional qualification of the medical specialists and the degree of availability of all necessary instrumentation specific for the respective disorders in the medical institution. 610 patient treated in the Neurosurgery Department of the Plovdiv University of Medicine between 1975 and 1990 were entered into this study. Three periods of the Department's diagnostic capabilities are differentiated within this interval. They are used as an analogue model for the assessment of diagnostic and therapeutic capabilities of the different types of hospitals. The patients are categorized as survivors and dead, diagnosed and nondiagnosed in the three periods. It is concluded that all methods available should be used in diagnosing traumatic intracranial hematomas. Their condition permitting patients should always be transported to a neurosurgery clinic where an optimal environment is provided for diagnostics and treatment. Employment of a portable echoencephalograph can additionally augment the diagnostic capabilities of the consultant neurosurgeon in small health-care units especially in cases in which transportation of the patient to a neurosurgery clinic is contraindicated.

A case of delayed subdural hematoma.

Over the recent years, a number of authors have assumed that traumatic intracranial hematomas reach their maximum size within minutes after the trauma. However, there are reports in the literature of proven traumatic intracranial hematoma of delayed onset after a negative computer tomographic or arteriographic study of the brain. We present a case of a 70-year-old patient admitted to the Clinic after a road accident. On admission the patient was disoriented in regard to time but his neurological status was otherwise normal. On the second day after admission the patient's condition deteriorated and he became comatose with moderate left hemiparesis. An emergency left carotid angiography was performed which was negative for a hematoma. Three days later the patient died. Postmortem examination disclosed a subdural hematoma in the left parietotemporal region with a volume of about 100 ml, as well as encephalomalacia and brain edema.

MR detection of quantitative and structural changes in human aortic aneurysms.

Collagen is a major component of the extracellular matrix and a determinant of the elastic behavior of the human aorta. To investigate the changes found in aneurysmal degeneration, the authors studied the solid-state hydrogen-1 nuclear magnetic resonance line shape of collagen in aneurysms and normal human aortas. A three-component decomposition of the free induction decay was performed, with collagen characterized by a T2 of about 18 microseconds. The second moment of the collagen line shape was found to be increased in aneurysms (5.3 vs 4.8 G2), while, correspondingly, the T2 of collagen was lower in aneurysms (16.3 vs 17.7 microseconds). This corresponds to a modification of collagen structure and molecular motion. Collagen concentration was lower in nondiseased aortic walls (9.4% vs 7.3%). These results are discussed in reference to the contradictory conclusions in the current literature. The increase in collagen and the modification of its structure and molecular motion are explained by the need to resist an increasing tangential tension due to increased aortic diameter and diminished wall thickness in aneurysms and by intercalation or site binding in the helices or electric dipolar interactions in the less mobile side groups.

Meningioma accompanied by a subdural hematoma. A case report. Review of 17 cases in the literature.

Hemorrhage occurs extremely rare in meningioma. We have found only 17 cases of meningioma associated with subdural hematoma in the reviewed literature. We report a similar case of a 26-year-old woman who complained initially of paralysis of her left arm evolving into loss of consciousness accompanied by a spasm of the extremities and foam in the mouth. In the days immediately preceding admission into hospital she complained of headache, unsteadiness in walking and vomiting. On admission she was bradypsychic, time-disoriented and drowsy. Latent left-sided hemiparesis was established. A right carotid angiography gave indication of a subdural hematoma. The patient was operated on in an emergency. After evacuation of the hematoma a smooth tumour was found embedded in the brain with a cyst in it. The tumour was the size of an egg, soft and elastic. There were several bleeding vessels underneath. During operation the tumour was not in contact with dura mater. The histologic diagnosis was fibroblastic meningioma. After treatment the patient was discharged in good condition with slight hemiparesis.

Characterization of human aortic collagen's elasticity by nuclear magnetic resonance.

The elasticity of the human aortic wall in longitudinal uniaxial elongation at high strain, known to be determined mostly from tissular collagen's behaviour, is studied and compared to the second moment of the 1H nuclear magnetic resonance (NMR) solid state line-shape, a proton nuclear magnetic resonance (at 60 MHz) characteristic for the molecular motion and the rigidity of the collagen macromolecular backbone. The 1H NMR signal of collagen is identified after selective histologically controlled chemical lysis. The computed second moment of the line-shape shows statistically significant correlation with the slope of the strain-stress curve of the aorta at high strain, thus proving the relationship between a macroscopic tissular elasticity parameter and a macromolecular rigidity characteristic of collagen, a major tissular component. In vivo extension of this technique (e.g., MRI) would allow us to gain information on the biomechanical state of the aorta, a naturally highly stressed and strained tissue.

Proton magnetic resonance relaxation times and biomechanical properties of human vascular wall. In vitro study at 4 MHz.

OBJECTIVES AND RATIONALE: Previous studies have suggested a relationship between tissue magnetic resonance (MR) relaxation times and its biomechanical behavior. To further investigate this relationship, the authors studied 41 human vascular wall samples from different anatomic localizations, including systemic and pulmonary arterial, as well as venous tissues. METHODS: The authors measured water content, proton MR T1 and T2 relaxation times, and two viscoelastic parameters of the samples at 4 MHz. RESULTS: T2, water content, and both viscoelastic variables significantly differed among the five anatomic localizations (P less than .05). Both T1 and T2 were significantly (P less than .05) and linearly related to viscoelastic parameters. Multiple linear regression showed that both viscoelastic parameters of a sample can be predicted from the measured values of T1 and T2. CONCLUSIONS: These results provide a basis for characterizing the mechanical stress of a tissue by knowing its MR relaxation times.

[Retrograde positive ventriculography by sub-occipital catheterization of the ventricular system (author's transl)]. / Retrograde positive Ventrikulographie durch subokzipitale Katheterisierung des Ventrikelsystems.

The authors describe a new method of retrograde selective ventriculography by means of which the necessity for making an opening in the skull and for a trans-cerebral ventricular puncture is avoided. Trough a sub-occipital puncture and cannula a radio-opaque catheter is guided under radiological control into the ventricular system.