Adenosine-deaminase-deficient mice die perinatally and exhibit liver-cell degeneration, atelectasis and small intestinal cell death.

We report the generation and characterization of mice lacking adenosine deaminase (ADA). In humans, absence of ADA causes severe combined immunodeficiency. In contrast, ADA-deficient mice die perinatally with marked liver-cell degeneration, but lack abnormalities in the thymus. The ADA substrates, adenosine and deoxyadenosine, are increased in ADA-deficient mice. Adenine deoxyribonucleotides are only modestly elevated, whereas S-adenosylhomocysteine hydrolase activity is reduced more than 85%. Consequently, the ratio of S-adenosylhomocysteine (AdoMet) to S-adenosyl homocysteine (AdoHcy) is reduced threefold in liver. We conclude that ADA plays a more critical role in murine than human fetal development. The murine liver pathology may be due to AdoHcy-mediated inhibition of AdoMet-dependent transmethylation reactions.

Human polyspecific immunoglobulin attenuates group A streptococcal virulence factor activity and reduces disease severity in a murine necrotizing fasciitis model.

OBJECTIVES: Streptococcus pyogenes causes life-threatening invasive infections including necrotizing fasciitis (NF). Current treatment guidelines recommend the use of a cell-wall-active antibiotic combined with a protein synthesis inhibitor and surgical debridement in NF patients. Adjunctive therapy with intravenous immunoglobulin (IVIG) has been proposed for superantigen-associated streptococcal toxic shock syndrome. So far, benefits of IVIG treatment remain unclear and prospective clinical studies are scarce. Thus, we aimed to assess the effects of IVIG on virulence factor activity in vitro, ex vivo in patients and in vivo in a NF mouse model. METHODS: We investigated the effect of IVIG on the activity of the virulence factors streptolysin O (SLO), streptodornase 1 (Sda1), S. pyogenes cell envelope protease and streptococcal pyrogenic exotoxin B in vitro and ex vivo in patient sera. Additionally, we assessed the influence of IVIG on the clinical outcome in a murine NF model. RESULTS: In vitro, IVIG inhibited various streptococcal virulence factors. Further, IVIG treatment of group A Streptococcus-infected mice led to a reduced skin lesion size (median (interquartile range) day 3 intraperitoneal administration: 12 mm2 (9-14.5) vs. 4 mm2 (0.8-10.5), subcutaneous: 10.3 mm2 (6.9-18.6) vs. 0.5 mm2 (0.1-6.8)) and lower SLO activity. After treatment with IVIG, patient sera showed an elevated titre of specific SLO (7/9) and Sda1 (5/9) antibodies, reducing SLO and Sda1 activity. CONCLUSIONS: The clear reduction in disease severity in IVIG-treated mice and inhibition of virulence factor activity in mouse and human sera suggest that IVIG may be beneficial in invasive group A Streptococcus infections such as NF in addition to streptococcal toxic shock syndrome.

Mdm2, but not Mdm4, protects terminally differentiated smooth muscle cells from p53-mediated caspase-3-independent cell death.

p53 is a potent inhibitor of cell growth and an inducer of apoptosis. During embryonic development, Mdm2 and Mdm4 inhibit the growth suppressive activities of p53. However, whether tight surveillance of p53 activity is required in quiescent cells is unknown. To test this, conditional inactivation of mdm2 and mdm4 was carried out in smooth muscle cells (SMCs). Upon SMC-specific inactivation of mdm2, and not of mdm4, mice rapidly became ill and died. Necropsy showed small intestinal dilation, and histological analyses indicated a severe reduction in the number of intestinal SMCs. Increased p53 levels and activity were detected in the remaining SMCs, and the phenotype was completely rescued on a p53-null background. Interestingly, intestinal SMCs are caspase-3-negative and therefore did not undergo caspase-3-dependent apoptotic cell death. Together, Mdm2, but not Mdm4, prevents accumulation of active p53 in quiescent SMCs and thereby the induction of p53-mediated caspase-3-independent cell death.

Induction of lung tumors by radioactive isotopes implanted in the rat lung.

Squamous cell carcinomas were induced in the lungs of male WAG/Rij inbred rats by radiation emitted from the isotopes iridium-192 or iodine-125. These isotopes were implanted by a surgical procedure in the lungs of young rats. Forty rats received implants of 192Ir wires and 20 animals, of 125I seeds. In a 14-month observation period, 30 of the 40 animals with implants of 192Ir wires developed tumors. Malignant hemangioendotheliomas occurred with the highest frequency (50%). From the lungs of 12 rats, squamous cell carcinomas were found. In the observation period of 17 months, 3 rats with implants of 125I seeds developed tumors, among which 1 squamous cell carcinoma could be identified. Tumor fragments were transplanted in syngeneic hosts for propagation of the tumors. Histologic appearances of tumors remained constant in subsequent passages. Responses of transplanted tumors growing in the flanks of syngeneic hosts to doses of radiation, methotrexate, or vinblastine were determined. Although the histologic appearances of the 5 squamous cell carcinomas were similar, tumor-doubling times and responses to irradiation and chemotherapeutic drugs were different. Small cell or large cell carcinomas were not observed.

Differential behavior of human bronchial carcinoma cells in culture.

A feeder layer culture system suited to grow carcinoma cells derived from solid human lung tumors was developed. This report deals with culturing of the four main histological types of lung carcinomas observed in 37 patients: 19 squamous cell, 6 adenocarcinomas, 7 small cell, and 5 large cell carcinomas. The cultures were initiated from 24 fresh human surgical specimens and from 14 human lung tumors grown as xenografts in nude mice. Three different patterns of behavior in culture were found to be characteristic for squamous cell, adenocarcinomas, and small cell carcinomas, respectively. The culture pattern presented by the primary cultures did not appreciably change after passaging in vitro for periods of up to 2 years, even after infinite cell lines were established. Cultures of large cell carcinoma showed one or more of these patterns. From these patterns cells could be cloned and subsequently cultured as separate stable lines. The system described facilitates the identification of specific types of human lung carcinomas almost immediately (within 1 h) after plating (Phase I) as well as during culture.

Effects of acute graft-vs-host disease on the liver of the brown Norway rat.

In this study we examined the effects of acute graft-vs-host disease (aGVHD) on the Brown Norway (BN) rat liver. When clinical signs of the disease appeared, rats were inoculated with fluorescent latex beads and 30 min later nonparenchymal cells were isolated from the liver. The cells were then analyzed via flow cytometry, histochemistry, and electron microscopy. Flow cytometry demonstrated that 58% of the cells from the 80 ml/min elutriation fraction (normally rich in Kupffer cells) of the non-GVHD liver had high fluorescence intensity compared to 8% in rats with aGVHD. Determination of the cellular composition of the various fractions with electron microscopy confirmed flow cytometry observations in that only 9% of the 80 ml/min elutriation fraction of GVHD livers had peroxidase-positive rough ER and the morphological appearance of macrophages as compared to 60% in the non-GVHD liver. The low percentage of fluorescent-positive Kupffer cells in the 80 ml/min elutriation fraction of the GVHD liver is attributed to a massive lymphocytic invasion of the liver and not necessarily to a defect in the mononuclear phagocyte system.

Gastrointestinal decontamination of dogs treated with total body irradiation and bone marrow transplantation.

Procedures for total and selective gastrointestinal decontamination of dogs are described. The selective procedure removed only Gram negative aerobic bacteria, yeast and fungi. Dogs receiving total decontamination were less susceptible to the GI syndrome following total body irradiation (TBI) than dogs receiving conventional care. After TBI and allogeneic bone marrow transplantation, serum albumin levels decreased in conventional animals, but remained normal in totally or selectively decontaminated animals. Exogenous infections occurred frequently in both irradiated, and totally decontaminated animals, but were absent in selectively decontaminated animals. Endogenous infections after total body irradiation were prevented only by total decontamination. Endogenous infections occurred in selectively decontaminated animals, but with milder clinical symptoms than in conventional animals. Appearance of donor type leukocytes and serum gamma globulin was slower in decontaminated animals than in conventionally treated controls. Acute graft versus host disease caused by a limited number of lymphocytes of a DLA identical littermate donor were prevented by selective gastrointestinal decontamination. Complications due to late immune reconstitution obscured the effect of decontamination on delayed graft versus host disease.

Evidence for a diminished maturation of preosteoblasts into osteoblasts during aging in rats: an ultrastructural analysis.

Bone is subject to continuous remodeling throughout life. The age-related loss of (trabecular) bone, leading to senile osteopenia, is mainly due to impaired bone formation. Osteoblasts (OB) and osteoclasts (OC) have been identified as playing a crucial role in the process of bone turnover, but the contribution made by their precursors is not well documented. We analyzed the cells of the osteoblast and osteoclast cell lineage along the trabecular bone of tibiae and the stromal cells in the marrow of aging BN/Bi Rij rats using electron microscopy. It appeared possible to distinguish preosteoblasts (pre-OB), OB, preosteoclasts (pre-OC), OC, and inactive bone-lining cells. Periods of increase, the maximal peak, and the decrease in trabecular bone volume were defined by means of morphometric measurements of trabecular bone volume. We found a decrease of more than 10-fold in the number of OB with age, but the numbers of pre-OB, pre-OC, and OC expressed per unit bone length, although variable, were age independent. The relative bone resorption and formation surface, expressed as a percentage of the total bone surface, decreased 2- and 15-fold, respectively. In 2-year-old animals the total volume of stromal cells, part of which constitutes the stem cell compartment of the osteogenic lineage, was a quarter of that found in 1-month-old animals and a third of that found in 6-month-old animals. The loss of trabecular bone is concomitant with a sharp increase in the ratio of pre-OB/OB, the ratio of OC/OB, and in the ratio of resorption to formation surfaces. There was no relation between the ratio of pre-OC/OC with age. These data lead to the conclusion that the main factor causing bone loss with age is a diminished maturation of pre-OB into OB.

Aerosol-mediated delivery of recombinant adenovirus to the airways of nonhuman primates.

At present, it is conceivable that gene therapy of the cystic fibrosis airway epithelium is possible using the direct transfer of a functional human cystic fibrosis transmembrane conductance regulator (CFTR) gene to a wide variety of patients' tracheo-bronchial cells. Here we describe a novel approach (aerosolization) to deliver a replication-deficient adenovirus carrying the CFTR gene (Ad.CFTR) to the airways. Results obtained in vitro and in Rhesus monkeys suggest that the delivery of recombinant adenovirus as an aerosol is feasible and is not associated with severe toxicity after single or double administration depending on the Ad.CFTR dose. This study supports the concept of aerosolization as a delivery method for adenovirus-mediated lung gene therapy.

Maintenance of donor phenotype after full-thickness skin transplantation from mice with chronic proliferative dermatitis (cpdm/cpdm) to C57BL/Ka and nude mice and vice versa.

Chronic proliferative dermatitis is a spontaneous mutation in C57BL/Ka mice (cpdm/cpdm) and is characterized by epithelial hyperproliferation, infiltration by eosinophils and macrophages, and vascular dilatation. To elucidate whether these pathologic features are the result of a local (skin) process or a consequence of a systemic disorder, transplantations were performed of full-thickness grafts of affected skin from cpdm/cpdm mice and normal skin from control (C57BL/Ka) mice on the back of cpdm/cpdm, C57BL/Ka and athymic nude mice. After 3 months, the grafts maintained the histologic phenotype of the donor animal. Intercellular adhesion molecule-1 continued to be expressed by basal keratinocytes of the cpdm/cpdm grafts after transplantation. In contrast, the basal keratinocytes of the C57BL/Ka grafts onto cpdm/cpdm mice remained negative for intercellular adhesion molecule-1 3 months after transplantation. An increased number of proliferating keratinocytes was present in the cpdm/cpdm skin-graft transplanted to nudes or to C57BL/Ka mice based on short-term bromodeoxyuridine labeling. The bromodeoxyuridine incorporation in the keratinocytes of the control C57BL/Ka skin grafts transplanted to cpdm/cpdm, nude, or C57BL/Ka mice was the same as in the keratinocytes of normal C57BL/Ka mice. This study demonstrates that the pathologic features found in the cpdm/cpdm mice are the result of a disorder in the epidermis or dermis and not due to a systemic defect.

The effect of aging on the hepatic metabolism of sulfo-bromophthalein in BN/Bi female and WAG/Rij male and female rats.

The effect of aging on sulfobromophthalein (BSP) metabolism was studied in three groups of rats-BN/Bi female and WAG/Rij male and female rats-of different ages ranging from 3 to 30 months. Under Nembutal anesthesia, BSP biliary transport maximum (Tm) and relative storage capacity (S) were determined by a single infusion rate method by directly determining Tm from bile samples collected through a common bile duct cannula. Tm values expressed as micrograms of BSP per min per g of liver were highest in the youngest rate (3-month-old) as compared with the older rats (12-, 24-, 30-month-old) for all three rat groups. Tm gradually decreased as age increased and at the age of 24 or 30 months reached a value of 66 - 70% of the highest values for 3-month-old rats. The percentage of conjugated BSP in the bile measured during the Tm period remained essentially unchanged with age in all three rat groups. S values, expressed as mg of BSP stored per mg or BSP per ml of plasma per g of liver, remained unchanged (BN/Bi female) or even increased (WAG/Rij male and female) with age. As a consequence, S values expressed per rat were higher in older age groups than in the youngest one for all three rat groups. In contrast with previous reports by other authors on man and rats, the BSP Tm appears to decrease with age regardless of rat and sex, while S does not show such a decrease.

Biological and clinical consequences of longitudinal studies in rodents: their possibilities and limitations. An overview.

Rats and mice are used in gerontological research primarily because of their relatively short life spans, ease of handling, and the relatively low costs of production and maintenance under controlled environmental conditions of large number of rodents as compared to larger laboratory animal species. They are being used as models for studying intrinsic aging processes, processes that give rise to diseases associated with aging, and the influence of environmental factors on these processes. Contrary to the situation in man, longitudinal studies in rodents can be conducted under well controlled environmental conditions. It has been shown that multiple pathology, the hallmark of aging in man, also occurs in inbred strains of rodents. Some of these lesions are genetically determined and some of them are randomly distributed amongst members of the same inbred strain. Serial killing experiments are necessary to obtain information on the time of development of these lesions in order to interpret properly the outcome of investigations. Furthermore, it has been shown that a considerable variation can exist in the observed maximum ages of the longest-lived animals in cohorts of rats kept under well controlled conditions. For this reason, caution should be exercised in interpreting data from studies which claim maximum lifespan prolongation.

Lung damage following bone marrow transplantation: II. The contribution of cyclophosphamide.

The effect of high-dose cyclophosphamide (Cy), either alone or in combination with irradiation, upon the development of interstitial pneumonitis (IP) after bone marrow transplantation (BMT) was investigated in a Brown Norway rat model. The parameters that were examined included ventilation rate, mortality, and histopathology. No damage to the lungs was observed in rats given Cy alone in supralethal dosages plus BMT, and mortality resulted from severe aplasia of hemopoietic and lymphoid tissues with multifocal hemorrhages, secondary infections, and sepsis. Two separate periods of mortality were observed within the first 180 days following whole thorax irradiation with a high dose rate (HDR; 0.8 Gy/min) or a low dose rate (LDR; 0.05 Gy/min). The addition of Cy prior to irradiation resulted in an increased mortality in the first period (before day 100) in all experimental groups. The influence of Cy on mortality at 180 days however, was different for the HDR and LDR experiments. The LD50-180 after HDR irradiation, dose range 8 to 18 Gy, was not significantly altered by the addition of Cy (100 mg/kg) 1 day prior to irradiation, whereas Cy (100 mg/kg) 1 day prior to LDR irradiation, dose range: 16 to 24 Gy, caused an enhancement of radiation damage with a decrease of the LD50-180 by 1.33 Gy. The dose modification factor (DMF) was 1.07. This enhancement was no longer significant after splitting up the dose of Cy in two dosages of 50 mg/kg given on 2 consecutive days prior to irradiation with a LDR. The extrapolation of the data in this rat model to available dose-response curves on IP after BMT and radiation pneumonitis in humans, implied that non-infectious IP is a radiation pneumonitis that is only slightly enhanced by Cy.

Lung damage following bone marrow transplantation: I. The contribution of irradiation.

High dose whole body irradiation is commonly included in conditioning regimens for bone marrow transplantation for treatment of patients with hematological malignancies. Interstitial pneumonitis is a major complication after BMT. About one-fourth of all BMT patients die from IP. In about half of these cases, an infectious agent, particularly cytomegalovirus, is involved. When no infectious cause is found, it is classified as idiopathic IP (IIP). Total body irradiation is often associated with the induction of IIP; however, extrapolation of animal data from the experiments presented indicates that this is not the only factor contributing to IIP in man. Brown Norway (BN/Bi) rats were bilaterally irradiated to the lungs with 300 kV X rays at a high dose rate (HDR; 0.8 Gy/min) and at a low dose rate (LDR; 0.05 Gy/min). The dose-response curves found were very steep. In the LDR group, lung function studies were performed. There was a strong correlation between the increase in ventilation rate and the death pattern. The LD50 at 180 days was 13.3 Gy for HDR and 22.7 Gy for LDR. The ratios of LD50/180 at 0.05 Gy/min to that at 0.8 Gy/min is 1.7, which indicates a great repair capacity of the lungs. Extrapolation of animal data to patient data leads to an estimated dose of about 15-16 Gy at a 50% radiation pneumonitis induction for low dose rate TBI. As the absorbed dose in the lungs of BMT patients rarely exceeds 10 Gy, additional factors such as remission-induction chemotherapy, cyclophosphamide, methotrexate, cyclosporin A, graft-versus-host disease, etc., might be involved in the high incidence of IIP in man after BMT.

OKT4 and OKT4A antibody treatment as immunosuppression for kidney transplantation in rhesus monkeys.

A mixture of OKT4+4A monoclonal antibodies (reactive with T4 cells) was tested for its immunosuppressive potential in rhesus monkeys receiving a kidney allograft. The kidney transplant model used in this study was designed to mimic the clinical situation. Therefore, all animals received a low dose of azathioprine and prednisolone, and the effectiveness of monoclonal antibody treatment was assessed in nontransfused and transfused recipients. The treatment very effectively suppressed acute graft rejection in untransfused recipients. In transfused recipients, which show an improved graft survival, no additional favorable effect of OKT4 + 4A treatment was seen when this treatment was given at the time of transplantation. It is possible that transfused recipients that reject their kidney in an acute fashion do not benefit from the OKT4 + 4A treatment because they have generated primed effector cells that belong to a T4-negative subpopulation or a T4-positive subpopulation with high affinity for donor cells. When the OKT4 + 4A treatment was given at the time of graft rejection in transfused recipients, thus treating chronic rather than acute rejection, a modest improvement in graft survival was observed. It seems, therefore, that anti-T4 antibodies will be of limited value for clinical transplantation and should be used in combination with other immunosuppressive drugs.

Engraftment of stem-cell-enriched bone marrow fractions in MHC-identical dogs after fractionated total-body irradiation.

Discontinuous albumin density gradients were used to obtain enrichment of hemopoietic stem cells and depletion of T lymphocytes in aspirated dog bone marrow. Colony forming units in agar (CFU-C) were determined to evaluate the degree of enrichment achieved. An average CFU-C concentration factor of 12.4 was obtained. All transplantations in the study were carried out between DLA-identical sibling combinations. The number of CFU-C administered varied from 0.2 to 5.5 X 10(5)/kg and the number of nucleated cells transfused varied from 0.1 to 1.0 X 10(8)/kg. Stem cell concentrates were found more difficult to engraft than unmodified bone marrow following standard conditioning with a single total-body irradiation (TBI) dose of 7.5 Gy. The efficacy of different TBI-fractionation schedules for obtaining sustained engraftment of CFU-C-enriched grafts in identical bone marrow transplantation (BMT) was determined. A total dose of 12 Gy TBI delivered in two equal fractions of 6.0 Gy (72-hr interval) resulted in sustained engraftment of stem cell grafts in 7 of 7 evaluable dogs. A TBI dose of 9 Gy in two fractions of 4.5 Gy (72-hr interval) resulted in sustained engraftment in 5 of 7 evaluable dogs. The two dogs with engraftment failure received low total cell numbers (10(7) cells/kg) and low CFU-C numbers. 9 Gy of TBI in two fractions of 4.5 Gy (24-hr interval) resulted in sustained engraftment in 11 of 12 evaluable dogs. A significant improvement of engraftment was obtained by increasing the total dose of TBI, which necessitates fractionation into two fractions of TBI. The lower-total-dose TBI (9 Gy) produced less early and late toxicity than the total high-dose (12 Gy) TBI. The incidence of engraftment was similar for the two dosages, however the recovery of peripheral leukocyte counts was slower after 9 Gy TBI. In the dog, optimal conditioning for lymphocyte-depleted hemopoietic stem cell grafts can be obtained by increasing the dose of TBI and concomitant fractionation.

Lasting engraftment of histoincompatible bone marrow cells in dogs.

Conditioning protocols were tested for their efficacy in increasing the incidence of engraftment of histoincompatible dog bone marrow cells. Cyclophosphamide and total body irradation (TBI), Corynebacterium parvum and TBI, a 3- or 5-day delayed transfusion of bone marrow cells after TBI, or an increase in the number of donor bone marrow cells or lymphocytes appeared to be ineffective. These protocols were previously reported to promote recovery of splenic hemopoiesis in mice in short-term assays. The noted discrepancy between studies with mice and dogs invalidated allogeneic resistance as measured in the mouse spleen assay as a model for bone marrow allograft rejection. Intravenous treatment with silica particles or L-asparaginase did improve the engraftment rate after 7.5 Gy TBI. Low efficiency and significant extra toxicity restrict the applicability of these procedures. The most promising conditioning schedule found appeared to be two fractions of 6.0 Gy TBI separated by a 72-hr interval. Prolonged survival was noted after transplantation of bone marrow cells from a one-DLA haplo-type-mismatched donor. Possibilities for further improvement of this protocol are discussed.

Thromboembolism and bleeding tendency in congenital factor XII deficiency--a study on 74 subjects from 14 Swiss families.

In order to assess the clinical implications of hereditary F XII deficiency, all available members of Swiss families with F XII deficiency were investigated. Based on the F XII:C values and the family pedigree, the 74 subjects, aged 8-82 years, were classified as homozygotes/double heterozygotes for F XII deficiency (n = 18), as obligatory (n = 20) or possibly (n = 25) heterozygotes, respectively, and as normals (n = 11). None of the 18 subjects with F XII:C less than 0.01 U/ml and only one possibly heterozygous woman had an abnormal bleeding tendency, confirming the notion that Hageman trait generally does not result in a hemorrhagic diathesis. Two of the 18 subjects with severe F XII deficiency had suffered from venous thromboembolic disease at age less than 40 years. One heterozygous woman had a leg ulcer probably due to venous thrombosis. Thus, whereas homozygous F XII deficiency may be associated with an increased risk for venous thromboembolic disease, partial F XII deficiency is not, by itself, a strong risk factor for thrombosis. Whereas 17 of the 18 subjects with F XII:C less than 0.01 U/ml had no detectable F XII:Ag, one cross reacting material-positive F XII deficient subject (F XII:Ag = 0.11 U/ml) was identified. The dysfunctional F XII, present in this subject's plasma and tentatively called F XII Bern, is the fourth abnormal F XII molecule identified so far.

HLA-B27 as a relative risk factor in ankylosing enthesopathy in transgenic mice.

HLA-B27 is a risk factor for several human diseases through a mechanism that is not yet understood. This article describes a naturally occurring joint disease in laboratory mice, ANKENT. ANKENT begins with mild inflammation and culminates in irreversible stiffening of the ankle and/or tarsal joints in one or both hind paws. The macroscopic and histologic features of ANKENT, its relationship to age, gender, and environment, and some immunologic aspects are considered. With respect to genetics, it is demonstrated that an HLA-B27 transgene is a relative risk factor for ANKENT. Its impact depends on the H-2 haplotype, reaching a relative risk value of 9.4 for C57Bl/10, H-2b males (p < 0.025). Several features of ANKENT are reminiscent of human AS: joint pathology, age and gender distribution, the presence of non-MHC as well as MHC risk factors (including HLA-B27), and the suspicion that environmental factors are involved.

A rat lung cancer model based on intrapulmonary implantation of tumour material.

A lung cancer model based on implantation of tumour fragments in the posterior lobe of the right lung of WAG/Rij rats by a surgical procedure has been developed. Two transplantable squamous cell carcinomas which were induced by implanted iridium-192 wires in the lung, were used in this study. The characteristics of these tumours following intrapulmonary implantation are reported with respect to growth rate, invasiveness and metastasizing capacity. The tumour volumes are determined by X-ray chest radiographs. Mean tumour volume doubling times are about 9 days. This system provides a means to evaluate treatment regimens involving irradiation and chemotherapeutic drugs.