Liver-on-chip model and application in predictive genotoxicity and mutagenicity of drugs.

Currently, there is no test system, whether in vitro or in vivo, capable of examining all endpoints required for genotoxicity evaluation used in pre-clinical drug safety assessment. The objective of this study was to develop a model which could assess all the required endpoints and possesses robust human metabolic activity, that could be used in a streamlined, animal-free manner. Liver-on-chip (LOC) models have intrinsic human metabolic activity that mimics the in vivo environment, making it a preferred test system. For our assay, the LOC was assembled using primary human hepatocytes or HepaRG cells, in a MPS-T12 plate, maintained under microfluidic flow conditions using the PhysioMimix® Microphysiological System (MPS), and co-cultured with human lymphoblastoid (TK6) cells in transwells. This system allows for interaction between two compartments and for the analysis of three different genotoxic endpoints, i.e. DNA strand breaks (comet assay) in hepatocytes, chromosome loss or damage (micronucleus assay) and mutation (Duplex Sequencing) in TK6 cells. Both compartments were treated at 0, 24 and 45 h with two direct genotoxicants: methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS), and two genotoxicants requiring metabolic activation: benzo[a]pyrene (B[a]P) and cyclophosphamide (CP). Assessment of cytochrome activity, RNA expression, albumin, urea and lactate dehydrogenase production, demonstrated functional metabolic capacities. Genotoxicity responses were observed for all endpoints with MMS and EMS. Increases in the micronucleus and mutations (MF) frequencies were also observed with CP, and %Tail DNA with B[a]P, indicating the metabolic competency of the test system. CP did not exhibit an increase in the %Tail DNA, which is in line with in vivo data. However, B[a]P did not exhibit an increase in the % micronucleus and MF, which might require an optimization of the test system. In conclusion, this proof-of-principle experiment suggests that LOC-MPS technology is a promising tool for in vitro hazard identification genotoxicants.

Impact of the filling status of the bladder and rectum on their integral dose distribution and the movement of the uterus in the treatment planning of gynaecological cancer.

PURPOSE: Determination of the impact of the filling status of the organs at risk (bladder and rectum) on the uterus mobility and on their integral dose distribution in radiotherapy of gynaecological cancer. METHODS: In 29 women suffering from cervical or endometrial cancer two CT scans were carried out for treatment planning, one with an empty bladder and rectum, the second one with bladder and rectum filled. The volumes of the organs at risk were calculated and in 14 patients, receiving a definitive radiotherapy, the position of the uterus within the pelvis was shown using multiplanar reconstructions. After generation of a 3D treatment plan the dose volume histograms were compared for empty and filled organs at risk. RESULTS: The mobility for the corpus uteri with/without bladder and rectum filling was in median 7 mm (95%-confidence interval: 3-15 mm) in cranial/caudal direction and 4 mm (0-9 mm) in posterior/anterior direction. Likewise, cervical mobility was observed to be 4 mm (-1-6 mm) mm in cranial/caudal direction. A full bladder led to a mean reduction in organ dose in median from 94-87% calculated for 50% of the bladder volume (P < 0.05, Wilcoxon's matched-pairs signed-ranks test). For 66% of the bladder volume the dose could be reduced in median from 78 to 61% (P < 0.005) and for the whole bladder from 42 to 39% (P < 0.005), respectively. No significant contribution of the filling status of the rectum to its integral dose burden was noticed. CONCLUSIONS: Due to the mobility of the uterus increased margins between CTV and PTV superiorly, inferiorly, anteriorly and posteriorly of 15, 6 and 9 mm each, respectively, should be used. A full bladder is the prerequisite for an integral dose reduction.

A test of behavioral family therapy to augment exposure for combat-related posttraumatic stress disorder.

This study tested a family-based skills-building intervention in veterans with chronic combat-related posttraumatic stress disorder (PTSD). Veterans and a family member were randomly assigned to 1 of 3 conditions: (a) waiting list, (b) 18 sessions of twice-weekly exposure therapy, or (c) 18 sessions of twice-weekly exposure therapy followed by 16 sessions of behavioral family therapy (BFT). Participation in exposure therapy reduced PTSD positive symptoms (e.g., reexperiencing and hyperarousal) but not PTSD negative symptoms. Positive symptom gains were maintained at 6-month follow-up. However, participation in BFT had no additional impact on PTSD symptoms.

[Comparison of different 3-dimensional irradiation techniques in local radiotherapy of prostatic carcinoma]. / Vergleich unterschiedlicher dreidimensionaler Bestrahlungstechniken bei der lokalen Strahlentherapie des Prostatakarzinoms.

PURPOSE: Four different three-dimensional planning techniques for localized radiotherapy of prostate cancer were compared with regard to dose homogeneity within the target volume and dose to organs at risk, dependent upon tumor stage. PATIENTS AND METHODS: Six patients with stage T1, 7 patients with stage T2 and 4 patients with stage T3 were included in this study. Four different 3D treatment plans (rotation, 4-field, 5-field and 6-field technique) were calculated for each patient. Dose was calculated with the reference point at the isocenter (100%). The planning target volume was encompassed within the 95% isodose surface. All the techniques used different shaped portal for each beam. Dose volume histograms were created and compared for the planning target volume and the organs at risk (33%, 50%, 66% volume level) in all techniques. RESULTS: The 4 different three-dimensional planning techniques revealed no differences concerning dose homogeneity within the planning target volume. The dose volume distribution at organs at risk show differences between the calculated techniques. In our study the best protection for bladder and rectum in stage T1 and T2 was achieved by the 6-field technique. A significant difference was achieved between 6-field and 4-field technique only in the 50% volume of the bladder (p = 0.034), between the 6-field and rotation technique (all volume levels) and between 5-field and rotation technique (all volume levels). In stage T1, T2 6-field and 4-field technique in 50% (p = 0.033) and 66% (p = 0.011) of the rectum volume. In stage T3 a significant difference was not observed between the 4 techniques. The best protection of head of the femur was achieved by the rotation technique. CONCLUSION: In the localized radiotherapy of prostate cancer in stage T1 or T2 the best protection for bladder and rectum was achieved by a 3D-planned conformal 6-field technique. If the seminal vesicles have been included in the target volume and in the case of large planning target volume other techniques should be taken for a better protection for organs at risk e. g. a 3D-planned 4-field technique box technique.

Viral load, CD4 percentage, and delayed-type hypersensitivity in subjects receiving the HIV-1 immunogen and antiviral drug therapy.

Two trials of subjects inoculated with the inactivated, gp120-depleted HIV-1 Immunogen are reported. In one study, in which 19 subjects received ZDV and 8 subjects received ddI, treatment with the HIV-1 Immunogen did not affect the pharmacokinetic parameters of the antiviral drugs. In another study, 65 subjects who were previously immunized with the HIV-1 Immunogen over a mean period of 4.0 years (range, 1.2-5.4 years) received inoculations at 0 and 6 months. At some point during this 48-week study, 72% of the subjects (47/65) were receiving antiviral drug therapy. The HIV-1 DNA load in CD4 cells and CD4 percentage were found to be stable over the 48-week period. Delayed-type hypersensitivity to HIV-1 antigens increased after two inoculations with the HIV-1 Immunogen. In these two trials, no serious treatment-related adverse events were documented in the subjects. The two studies presented herein are the first to suggest that an immune-based therapy such as the HIV-1 Immunogen can be combined safely with antiviral drugs, supporting further study to evaluate the clinical utility of this approach.

Initial studies on active immunization of HIV-infected subjects using a gp120-depleted HIV-1 Immunogen: long-term follow-up.

In 1987, exploratory clinical studies were initiated to determine whether the development of AIDS in HIV-infected individuals might be delayed or prevented by immunization with an inactivated HIV preparation. Preclinical studies had shown the preparation to be safe and immunogenic. Twenty-three patients with biopsy-confirmed persistent generalized lymphadenopathy (CDC III) and two with asymptomatic HIV infection and CD4 lymphocyte counts between 135 and 769/mm3 were studied, of whom eight (32%) had additional HIV-related symptoms. Over a 3-year period, they received a median of eight open-label inoculations of 100 micrograms of inactivated gp 120-depleted HIV-1 Immunogen in incomplete Freund's adjuvant (IFA). Clinical, general laboratory, immunologic, and virologic parameters were followed for up to 6 years. No serious treatment-related adverse experiences were reported, nor was accelerated HIV disease progression seen. Twelve patients developed a delayed-type hypersensitivity response (HIV-DTH) to the immunogen and nine showed fourfold or greater increases in anti-p24 antibody titers. In the follow-up period, 10 of the 25 patients developed AIDS and one with Kaposi's sarcoma (KS) at baseline progressed. Of the 12 patients who became HIV-DTH-responsive, one developed an opportunistic infection (OI), occurring approximately 5 years from study onset, and subsequently died. One additional HIV-DTH responder developed KS. Of the 13 patients who remained HIV-DTH-nonresponsive, nine (69%) progressed to AIDS and seven of these have died. Differences were also observed in terms of HIV-DNA copy number, CD4 percentages, and anti-p24 antibody patterns between the HIV-DTH-responsive and -nonresponsive groups, suggesting a more favorable clinical course in the former. HIV-1 Immunogen in IFA appears to be safe and immunogenic. Further studies are indicated to determine clinical efficacy of the HIV Immunogen as well as the significance of the apparent correlation between HIV-DTH responsivity and a more favorable clinical course.

Behavioral family therapy for Vietnam combat veterans with posttraumatic stress disorder.

Severe interpersonal problems are common in veterans with combat-related posttraumatic stress disorder (PTSD) and their families. The authors first detail the rationale and use of behaviorally based family therapy to help reduce avoidance and withdrawal symptoms of PTSD and improve the active coping capacities of both veterans and their loved ones, then present a case example of the model, and finally discuss the clinical application of behavioral family therapy to the unique concerns of veterans with combat-related PTSD.

Induction of humoral and cellular immunity to simian immunodeficiency virus: what are the requirements for protection?

In an effort to produce a strong humoral and cellular immune response that might protect against simian immunodeficiency virus (SIV) infection, groups of five rhesus macaques each were immunized intramuscularly at 0, 2 and 6 months with 100 micrograms of an inactivated preparation of SIV/Delta B670 in either an oil-in-water emulsion with Ribi Detox, containing mycobacterial cell wall skeleton and monophosphoryl lipid A (CWS/MPL) (group A) or a water-in-oil emulsion with incomplete Freund's adjuvant, containing CWS/MPL for the first two injections (group B). Animals were challenged with 10-100 monkey ID50 of monkey-cell-grown SIVmac251 3 months after the last injection, along with a group of four unvaccinated controls. Group B animals demonstrated the strongest immune responses following immunization, including neutralizing antibody titres against the challenge virus ranging from 160 to 320 and SIV-specific ELISA titres ranging from 10(5)-10(6) on the day of challenge, as well as strong in vitro lymphoproliferative and interleukin-2 (IL-2) production responses to the immunogen. Neutralizing antibody was not detectable in group A animals, ELISA titres were lower (10(2)-10(4)), no in vitro lymphoproliferative responses were observed, and in vitro IL-2 production was less pronounced. No protection against challenge was observed in either group. Moreover, group B animals exhibited a more pronounced clinical response following challenge than either group A animals or controls, consisting of hyperthermia and a greater degree of lymphadenopathy on day 7, followed by hypothermia and generally higher levels of serum viraemia on day 14.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of autoimmunity and AIDS: prospects for therapeutic intervention.

The network theory of autoimmunity is presented with recent experimental data relevant to the understanding of the pathogenesis of AIDS. Schematically, effector T cells specific for self-antigens exist normally, but their activity is modulated and prevented by networks of regulatory T cells. As a result of mimicry between molecular components of microorganisms and self-antigens, autoimmune disease can be triggered by specific foreign pathogens which alter the state of activity of the network from suppression to activation. Conversely, by a procedure known as T-cell vaccination, autologous effector T cells re-injected after in vitro stimulation and attenuation may alter the state of the network from an activation to a suppression. Numerous observations are reviewed that support the concept of autoimmune activity in the destruction of non-infected T4 cells. Such activity is presumed to be triggered by an antigen of viral origin, the most likely, but not the only one, being the envelope protein gp 120. Based on this hypothesis, a T-cell vaccination procedure against effector T cells responsible for autoimmunopathic activity in HIV-seropositive patients is proposed, similar to the one known from experimental study of autoimmunity and presently being tested in human autoimmune diseases. Its purpose would be to prevent T-cell loss and the onset of immunodeficiency disease in HIV-seropositive patients. Apart from its potential therapeutic value, this procedure will have use as a therapeutic test from which insight will be gained about the immunopathogenesis of AIDS.

Can AIDS be prevented by T-cell vaccination?

T-cell vaccination as a specific prophylactic and therapeutic procedure has been shown to be effective in animal models of autoimmune disease. As autoimmunopathogenic components have been implicated in HIV infection, the authors propose a therapeutic test utilizing T-cell vaccination and suggest that AIDS could be prevented by such a procedure.

Observations after human immunodeficiency virus immunization and challenge of human immunodeficiency virus seropositive and seronegative chimpanzees.

Two human immunodeficiency virus (HIV)-seropositive chimpanzees (A-3 and A-86c) infected 4 yr earlier with HIV, along with one uninfected animal (A-36), were inoculated intramuscularly three times in a year with a gamma-irradiated gp120-depleted HIV immunogen in incomplete Freund's adjuvant. Both previously infected animals promptly developed an anamnestic humoral antibody response after the first dose, and the uninfected animal developed a primary humoral response to the first dose and then an anamnestic response to the second dose. Although HIV had been recovered repeatedly from the seropositive animals, they became persistently virus-culture negative at the time of or just before the first inoculation of the immunogen. Intravenous challenge with 40 chimpanzee-infectious-doses of a heterologous HIV strain (HIVIIIB) was done 4 mo after the third inoculation in the three treated chimpanzees and in an untreated control animal (A-189a). The immunized naive animal (A-36) and the unimmunized control (A-189a) became infected, and virus has been isolated from their peripheral blood mononuclear cells for greater than 2 yr after challenge. However, the two previously infected chimpanzees (A-3 and A-86c) resisted challenge and have remained virus negative by peripheral blood mononuclear cell cocultivation for greater than 2 yr of observation after challenge; moreover, no evidence of reinfection was detectable by PCR. Despite the in vivo resistance, however, peripheral blood mononuclear cells from the resistant animals (A-3, A-86c) remained susceptible to infection by HIV in vitro. These findings reveal that a state of immunity can develop and/or be induced to control and/or prevent HIV infection in the chimpanzees. In the absence of any detectable level of neutralizing antibody in A-3 and a low level in A-86c, the patterns of the responses to challenge seen in the four animals suggest that the cell-mediated immune mechanism must have played a significant role in the resistant chimpanzees both in control of their HIV infection and in their resistance to challenge.

Are booster doses of poliovirus vaccine necessary?

In this issue of VACCINE, Böttiger points out that 'When the booster dose of polio vaccine at the age of 6 years was introduced (in Sweden) in the 1960s ... neither the necessity, nor the appropriate age for a booster had been scientifically evaluated'. If such a scientific evaluation were to be made now, what insights might emerge that would influence our thinking about booster doses and the associated implications for effective vaccination programmes and policy?

The effect of angiotensin-converting enzyme inhibition on prolactin responses in normal and hyperprolactinemic subjects.

Recent observations implicate angiotensin-II (AII) as a possible PRL-releasing factor. These observations prompted us to investigate the role of the renin-angiotensin system in PRL release in man. Nine normal volunteers ingesting a 20-40 mmol/day sodium, 70 mmol/day potassium diet and eight normal volunteers ingesting a 120 mmol/day sodium, 70 mmol/day potassium diet were infused with metoclopramide (2.5 mg over 1 min) and later with TRH (500 micrograms), two agents known to cause PRL release. The infusions were repeated after 36 h of oral administration of converting enzyme inhibitor [CEI; captopril (50 mg, orally, four times daily) or enalapril (5 mg, orally, twice daily)]. On a separate occasion, AII was infused at 10 ng/kg.h for 1 h into normal volunteers on normal salt diet. CEI administration lowered mean arterial pressure by 6-7 mm Hg and stimulated the release of active renin. The PRL responses on low and normal salt diet as well as before and after CEI were not statistically different. There was also no difference in the PRL responses of patients placed on captopril vs. those on enalapril. AII increased blood pressure by 11-25 mm Hg, but did not increase PRL significantly above basal concentrations during the control dextrose infusion. Five hyperprolactinemic volunteers were also given CEI for up to 4 weeks. They demonstrated no significant change in serum PRL levels. We conclude that AII in the pituitary does not significantly alter either basal PRL levels or metoclopramide- and TRH-induced PRL responses in normal subjects on low and high salt diets. In addition, CEI is not a useful therapy in patients with pathological hyperprolactinemia. These findings, however, do not exclude a role for AII in physiological regulation, since CEI does not cross the blood-brain barrier and would not be expected to alter hypothalamic AII.

Immunologic memory induced at birth by immunization with inactivated polio vaccine in a reduced schedule.

One hundred forty-one healthy newborns were immunized 24 hours after birth with one dose of inactivated polio vaccine (IPV) of enhanced potency. Following the administration of a second vaccine dose six months later, a considerable proportion of babies responded with neutralizing antibody (NA) to the three poliovirus types. The very rapid occurrence and high antibody titer were indicative of an anamnestic response. Twenty-one infants who still had NA less than 1:4 to one-more poliovirus types after the second vaccine dose responded with very high NA values 7-10 days after a supplementary dose of IPV. It appears that IPV of enhanced potency administered at birth is apt to induce immunologic memory, which should provide the basis for protection against paralytic poliomyelitis in case of exposure to wild poliovirus later in life.

NIH conference. Development and evaluation of a vaccine for human immunodeficiency virus (HIV) infection.

The development of a safe and effective vaccine for infection with human immunodeficiency virus (HIV) is complicated by several unique scientific, logistic, and ethical issues. These issues include a lack of understanding of protective immunity to HIV and disease development, the absence of an adequate and convenient animal model for studying HIV infection, and difficulties in phase III evaluation of candidate vaccines. Because HIV can be transmitted as either a cell-free or cell-associated virus, a protective immune response against HIV infection will likely require both humoral and cell-mediated immunity. A neutralizing antibody against HIV and an antibody involved in antibody-dependent cellular cytotoxicity have been shown in HIV-infected persons, but their precise relation to protection is unclear. Cytotoxic lymphocytes from HIV-infected persons have been shown to lyse target cells expressing HIV or its proteins. Cloned T cells have been developed that manifest HIV-specific, major histocompatibility-complex class I-restricted cytotoxic capabilities that are broadly specific. Thus far, all attempts to protect chimpanzees, currently the only suitable animal model, from HIV infection have failed. Ongoing vaccine studies in humans include phase I trials of recombinant proteins of the HIV envelope in uninfected persons as well as the administration of whole killed virus to persons already infected with HIV. Rapid progress is being made in the development of new animal models for HIV infection. The establishment of alternative animal models, both primate and small animal models, will greatly facilitate the development of a vaccine for HIV infection.