Different Routes Of Injection Research Articles

Extracellular vesicles engineered to bind albumin demonstrate extended circulation time and lymph node accumulation in mouse models.

Extracellular vesicles (EVs) have shown promise as potential therapeutics for the treatment of various diseases. However, their rapid clearance after administration could be a limitation in certain therapeutic settings. To solve this, an engineering strategy is employed to decorate albumin onto the surface of the EVs through surface display of albumin binding domains (ABDs). ABDs were either included in the extracellular loops of select EV‐enriched tetraspanins (CD63, CD9 and CD81) or directly fused to the extracellular terminal of single transmembrane EV‐sorting domains, such as Lamp2B. These engineered EVs exert robust binding capacity to human serum albumins (HSA) in vitro and mouse serum albumins (MSA) after injection in mice. By binding to MSA, circulating time of EVs dramatically increases after different routes of injection in different strains of mice. Moreover, these engineered EVs show considerable lymph node (LN) and solid tumour accumulation, which can be utilized when using EVs for immunomodulation, cancer‐ and/or immunotherapy. The increased circulation time of EVs may also be important when combined with tissue‐specific targeting ligands and could provide significant benefit for their therapeutic use in a variety of disease indications.

Analyzing the Role of Proteases in Breast Cancer Progression and Metastasis Using Primary Cells from Transgenic Oncomice.

It is becoming increasingly evident that progression and metastasis of solid cancers is driven by the interaction of oncogene-transformed cancer cells and non-malignant host cells in the tumor stroma. In this process, the immune system contributes a complex set of highly important pro- and antitumor effects, which are not readily recapitulated by commonly used xenograft cancer models in immunodeficient mice.Therefore, we provide protocols for isolation of primary tumor cells from the MMTV-PymT mouse model for metastasizing breast cancer and their resubmission to congenic immunocompetent mice by orthotopic transplantation into the mammary gland or different routes of injection to induce organ-specific experimental metastasis, including intravenous, intracardiac, and caudal artery injection of tumor cells. Moreover, we describe protocols for sensitive detection and quantification of the metastatic burden.

The Effect of Different Routes of Injection of Bone Marrow Mesenchymal Stem Cells on Parotid Glands of Rats Receiving Cisplatin: A Comparative Study

Background and ObjectivesCisplatin is a powerful antitumor chemotherapeutic agent that is widely used in the treatment of many cancers but it has many side effects on many organs including salivary glands. Bone marrow is considered to be a rich environment that comprises many types of stem cells of which BMSCs (Bone marrow mesenchymal stem cells) are the most studied with potentiality to differentiate into many cell types. This study was conducted to evaluate the effect of different routes of injection of BMSCs on parotid glands of rats receiving cisplatin.Methods and ResultsSprague-Dawley rats were divided into 3 groups: a negative control group receiving phosphate buffered saline, a positive control group receiving cisplatin, and an experimental group where rats received cisplatin and then received iron oxide-labeled BMSCs by either intravenous or intraparotid routes or both. Animals were sacrificed at periods of 3,6,10 and 15 days after cisplatin injection, then histological, ultrastructural and immunohistochemical studies were done. The experimental stem cell treated group showed better histological features and increased PCNA proliferation index when compared to the control. The systemic and combination groups showed better results than the local group. Iron oxide-labeled cells were detected with Prussian blue stain.ConclusionsThis study proved that BMSCs can improve cisplatin induced cytotoxicity in parotid glands. Systemic administration showed to have a better effect than local intraparotid administration and comparable effect to combined administration.

In vivo characterization of the immune response towards the pathogenic Escherichia coli antigen SslE and modulation of the intestinal microbiota

Abstract Pathogenic E. coli, both intestinal (InPEC) and extraintestinal (ExPEC), account for a wide range of diseases, which can be fatal, across developing and developed countries. The vast array of virulence factors they can acquire may dramatically increase the severity of the infection. Emergence of resistant strains often renders antibiotics inefficient; in the case of Shiga toxin-producing E. coli, antibiotics can even be detrimental. Hence, a broad spectrum E. coli vaccine could be a promising alternative to prevent the spread of such diseases, while offering the potential for covering against several InPEC and ExPEC at once. Using the «reverse vaccinology» approach on an ExPEC strain, nine antigens were identified as protective against a mouse sepsis model. With 82% protective efficacy, SslE (Secreted and Surface-associated Lipoprotein of E. coli) was the most potent candidate; additional models demonstrated that SslE was also cross-protective against other ExPEC strains. Functional assays have demonstrated in vitro and in vivo that SslE is a mucinase which plays an important role for colonization and virulence of E. coli. To better understand the mechanism behind such protection, particularly at the mucosa, we are investigating the intestinal immune response obtained after immunizations with SslE using different routes of injection. In-depth analysis of each regimen will allow us to determine the most efficient method of immunization and adjuvant choice for SslE to be protective. Further, using this model of immunization, we will evaluate the potential perturbation caused by SslE on a human intestinal microbiota. These findings will be important to help us optimize the development of a potential broad spectrum vaccine.

The Intravenous Route Optimizes Engraftment and Survival in the Murine Model of in Utero Hematopoietic Cell Transplantation (IUHCT)

IntroductionIUHCT has the potential to treat many congenital hematologic and immune disorders. It is a nonmyeloablative, nonimmunosuppressivetransplant approach that allows engraftment across immune barriers. Obtaining levels of engraftment necessary to treat target diseases remains a significant impediment to the clinical application of IUHCT. One barrier to consistent high-level engraftment is competition from host HSCs for limited niches at the time of transplant. The fetal liver (FL) is the hematopoietic organ in the mouse, large animals, and humans at the proposed time of IUHCT. Techniques that optimize donor cell delivery to the FL may enhance engraftment following IUHCT. In this respect, we sought to identify the route of injection that results in the least degree of mortality while promoting the highest level of engraftment. We compared the intravascular (IV), intraperitoneal (IP), and intrahepatic (IH) delivery of donor hematopoietic cells in a congenic murine model of IUHCT. We hypothesized that the IV route would promote direct homing to the FL resulting in increased levels of postnatal peripheral blood (PB) chimerism, and allow for larger cell doses without deleterious effects on fetal survival.MethodsFive million C57Bl/6-GFP bone marrow mononuclear cells were suspended in a fixed volume of PBS (5 uL) and injected into embryonic day 14 (E14) C57Bl/6 fetuses via the IV, IP, or IH route. Flow cytometry and fluorescence stereomicroscopy (FSM) were used to assess donor cell homing to and engraftment of the FL at 4, 24 and 72 hours and the fetal spleen (FS) at 72 hours post IUHCT. Long-term postnatal PB engraftment was assessed monthly by flow cytometry. Fetal survival to E18 following increasing volumes of PBS injectate at E14 was compared between the different routes of injection. Statistical analysis was performed using ANOVA with Bonferronicorrection and FisherÕs Exact Test.ResultsThe IV route led to significantly higher engraftment of the FL at 24 and 72 hours and the FS at 72 hours post IUHCT (Figure 1). FSM revealed important route-dependent differences in the patterns and kinetics of donor cell homing. IV injected donor cells robustly populated the FL in a homogenous manner within 4 hours of IUHCT. Most IP injected cells remained localized to the peritoneal cavity for over 24 hours post IUHCT with only modest engraftment of the FL. IH injected cells remained within the liver in a focal distribution with notable leaking into the perihepatic space beyond 24 hours post IUHCT. The increased efficiency with which donor cells homed to and engrafted the FL following IV IUHCT translated into superior and stable long-term donor cell engraftment (Figure 2). Fetal mice also demonstrated increased survival following higher injectate volumes administered via the IV route (Figure 3). Tolerance to larger volumes permitted higher donor cell doses to be administered at a given concentration (1x106 cells/mL), which translated into higher long-term donor cell engraftment (6 month chimerism, IV 5x106 donor cells: 4.8 +/- 0.5% vs. IV 20x106 cells: 13.2 +/- 1.0%; p < 0.05).ConclusionsIn a congenic murine model of IUHCT, the IV route of injection results in increased levels of stable long-term donor cell engraftment compared to the IH and IP routes. Mechanistically, this is related to the increased efficiency of donor cell homing to the site of the hematopoietic niche at the time of the transplant. Furthermore, the IV route allows for larger volumes of injectate, and thus potentially higher cell doses, with reduced fetal loss. These observations have important implications in the translation of IUHCT technique to future clinical trials. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

New Design Of Human T-ALL Transplantation In NSG Mice Uncovers The Major Role Of CD31/PECAM1 In The Central Nervous System Infiltration

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is mainly a child and adolescent blood malignancy. T-ALL patients present an increased risk of Central Nervous System (CNS) relapse defined by leukemic cell infiltration in cerebrospinal fluid and brain. Using transgenic mice and T-ALL cell lines previous works have shown that T-ALL migration in CNS depends on CCR7 chemokine receptor expression (S. Buonamici et al., Nature, 2009). VE-cadherin and CD31/PECAM1 also seem implicated, as it has been shown in vitro (S. M. Akers et al., Exp Hematol, 2010). In patients, high level of IL-15 at diagnosis predicts current CNS invasion and sometimes at relapse (G. Cario et al., J Clin Oncol, 2007). So far no study has investigated mechanisms involved in CNS infiltration using T-ALL patient samples in vivo. In the present study we developed a mouse model of CNS infiltration using leukemic cells isolated from patients and transplanted into NOD/SCID IL2Rуc-/- (NSG) mice. Proper conditioning of NSG mice and different routes of injection were tested to define a protocol avoiding non-specific CNS infiltration of leukemic cells. Also bone marrow (BM) engraftment levels of leukemia between 60 to 100% were used to set up the excision time of hematopoietic tissues and brain. Leukemic blasts from 8 patients with or 9 patients without CNS invasion were grafted and brain infiltration was followed up using standard histology and immunohistochemistry techniques. Our data indicate that (1) under specific experimental procedures, leukemic cells from patients with CNS invasion did infiltrate mouse CNS (8/8 samples) whereas the majority of cells from “non-infiltrated” patients did not (7/9 samples), (2) leukemic cells recovered from NSG brain and BM were similar in terms of brain and/or BM infiltration in secondary transplant experiments. Moreover, T-Leukemia Initiating Cell frequency was the same whatever the BM or CNS origin of blasts in the primary recipient. Interestingly, analysis of blasts at diagnosis showed that surface expression of adhesion molecules can not discriminate CNS+ or CNS- leukemic cells. However, blocking of CD31 decreased in vitro migration of blasts from CNS+ compared to CNS- patients through endothelial layer derived from blood brain barrier cells. Pioneered in vivo experiments show that CNS+ blasts pre-treated with CD31 antibody and injected in NSG are less prone to colonize mouse brain. Moreover, knocking down CD31 in CNS+ T-ALL by lentiviral shRNA strategy impairs leukemia development in mice, further decreasing CNS infiltration, whatever injection routes is used including intrafemoral injection. In conclusion, T-ALL xenografts in NSG mice mimic CNS invasion in patients. CD31 is a major player in blast cells migration in vitro and brain infiltration in vivo. This new model opens a new area of investigation to improve our knowledge of the molecular mechanisms of CNS infiltration in T-ALL. Disclosures: No relevant conflicts of interest to declare.

Protective and Anti-Pathology Effects of Sm Fructose-1,6-Bisphosphate Aldolase-Based DNA Vaccine against Schistosoma mansoni by Changing Route of Injection

This study aimed to evaluate the efficacy of fructose-1,6-bis phosphate aldolase (SMALDO) DNA vaccination against Schistosoma mansoni infection using different routes of injection. The SMALDO has been cloned into the eukaryotic expression vector pcDNA3.1/V5-His TOPO-TA and was used in injecting Swiss albino mice intramuscularly (IM), subcutaneously (SC), or intraperitoneally (IP) (50 µg/mouse). Mice vaccinated with non-recombinant pcDNA3.1 served as controls. Each group was immunized 4 times at weeks 0, 2, 4, and 6. Two weeks after the last booster dose, all mice groups were infected with 80 S. mansoni cercariae via tail immersion. At week 8 post-infection, animals were sacrificed for assessment of parasitological and histopathological parameters. High anti-SMALDO IgG antibody titers were detected in sera of all vaccinated groups (P<0.01) compared to the control group. Both the IP and SC vaccination routes resulted in a significant reduction in worm burden (46.2% and 28.9%, respectively, P<0.01). This was accompanied by a significant reduction in hepatic and intestinal egg counts (41.7% and 40.2%, respectively, P<0.01) in the IP group only. The number of dead eggs was significantly increased in both IP and IM groups (P<0.01). IP vaccination recorded the highest significant reduction in granuloma number and diameter (54.7% and 29.2%, respectively, P<0.01) and significant increase in dead miracidia (P<0.01). In conclusion, changing the injection route of SMALDO DNA vaccination significantly influenced the efficacy of vaccination. SMALDO DNA vaccination via IP route could be a promising protective and anti-pathology vaccine candidate against S. mansoni infection.

Acute toxicity of berberine and its correlation with the blood concentration in mice

The aim of this study was to investigate the LD 50 (median lethal dosage) of berberine (BBR) through three different routes of injection in mice: intravenous (IV) injection, intraperitoneal (IP) injection, and intragastric (IG) oral administration. The concentration of BBR in blood from their IG doses (10.4, 20.8, 41.6, and 83.2 g/kg) and the content relationship of BBR among different injections were analyzed by high-performance liquid chromatography (HPLC). The LD 50 of BBR from IV and IP injections is 9.0386 and 57.6103 mg/kg, respectively; but no LD 50 was found in the IG group. A significant difference in bioavailability was observed between the different routes. Furthermore, the concentration of BBR in the blood from different IG doses was also significantly different. However, we discovered an interesting phenomenon indicating that the absorption of BBR by oral administration has a limit, therefore, explaining the difficulty in obtaining an LD 50 of BBR for IG injection. From the analysis of BBR content in blood after various administrations, we hypothesized that not only does the concentration of BBR in blood contribute to its acute toxicity, but also the routes of administration may be an important facet that affects this toxicity evaluation.

SV40 Pseudovirion gene delivery of a toxin to treat human adenocarcinomas in mice

SV40 vectors packaged in vitro (pseudovirions) are an efficient delivery system for plasmids up to 17.7 kb, with or without SV40 sequences. A truncated Pseudomonas exotoxin gene (PE38) was delivered into various human cells (HeLa, KB-3-1, human lymphoblastoids, and erythroleukemia cells), in vitro using pseudovirions. The number of viable cells was reduced significantly in the PE38-transduced cells. Human KB adenocarcinomas growing in mice were treated with intratumoral injection of PE38 packaged in vitro, and tumor size decreased significantly. Intraperitoneal treatments were as effective in reducing tumor size as intratumoral treatments. To check the viability of mock- or PE38-treated mice, every 4 days they were weighed, their blood was tested, and various tissues were screened for pathology. All parameters showed that the in vitro-packaged vectors, injected into tumors or intraperitoneally, caused no abnormalities in mice. The combined treatment of doxorubicin with in vitro-packaged PE38 reduced tumor size slightly more than each of the treatments separately. However, the combined treatment did not cause the weight loss seen with doxorubicin alone. These results indicate that SV40 in vitro packaging is an effective system for cancer gene delivery using two different routes of injection and in combination with chemotherapy.

Cross-packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity.

The serotypes of adeno-associated virus (AAV) have the potential to become important resources for clinical gene therapy. In an effort to compare the role of serotype-specific virion shells on vector transduction, we cloned each of the serotype capsid coding domains into a common vector backbone containing AAV type 2 replication genes. This strategy allowed the packaging of AAV2 inverted terminal repeat vectors into each serotype-specific virions. Each of these helper plasmids (pXR1 through pXR5) efficiently replicated the transgene DNA and expressed helper proteins at nearly equivalent levels. In this study, we observed a correlation between the amount of transgene replication and packaging efficiency. The physical titer of these hybrid vectors ranged between 1.3 x 10(11) and 9.8 x 10(12)/ml (types 1 and 2, respectively). Of the five serotype vectors, only types 2 and 3 were efficiently purified by heparin-Sepharose column chromatography, illustrating the high degree of similarity between these virions. We analyzed vector transduction in reference and mutant Chinese hamster ovary cells deficient in heparan sulfate proteoglycan and saw a correlation between transduction and heparan sulfate binding data. In this analysis, types 1 and 5 were most consistent in transduction efficiency across all cell lines tested. In vivo each serotype was ranked after comparison of transgene levels by using different routes of injection and strains of rodents. Overall, in this analysis, type 1 was superior for efficient transduction of liver and muscle, followed in order by types 5, 3, 2, and 4. Surprisingly, this order changed when vector was introduced into rat retina. Types 5 and 4 were most efficient, followed by type 1. These data established a hierarchy for efficient serotype-specific vector transduction depending on the target tissue. These data also strongly support the need for extending these analyses to additional animal models and human tissue. The development of these helper plasmids should facilitate direct comparisons of serotypes, as well as begin the standardization of production for further clinical development.

Immunization with plasmid DNA encoding hepatitis C virus envelope E2 antigenic domains induces antibodies whose immune reactivity is linked to the injection mode

Plasmids expressing different domains of the hepatis C virus (HCV) envelope E2 glycoprotein from a genotype 1a isolate were constructed to compare the immunogenic potential of E2 in nucleic acid-based immunizations. One plasmid, pCIE2t, expressed a C-terminally truncated form of E2, while others, pS2.SE2A to pS2.SE2E, encoded the adjacent 60-amino-acid (aa) sequences of E2 (inserts A to E) expressed as a fusion with the hepatitis B virus surface antigen. BALB/c mice were given injections of the plasmids intramuscularly (i.m.) or intraepidermally (i.e.) via a gene gun (biolistic introduction), and induced humoral immune responses were evaluated. The i.e. injections resulted in higher seroconversion rates and antibody titers, up to 100-fold, than did the i.m. injections (P = 0.01 to 0.04). Three restricted immunogenic domains, E2A (aa 384 to 443), E2C (aa 504 to 555), and E2E (aa 609 to 674), that yielded antibody titers ranging from 1:59 to > 1:43,700 could be identified. Subtype 1a- and 1b-derived E2 antigens and synthetic peptides were used in Western blot and enzyme-linked immunosorbent assay analyses, which revealed that the cross-reactivity of the plasmid-induced antibodies was linked both to the type of antigen expressed and to the injection mode. Induced anti-E2 antibodies could immunoprecipitate noncovalent E1E2 complexes believed to exist on the surface of HCV virions. This study allowed us to identify restricted immunogenic domains within E2 and demonstrated that different routes of injection of HCV E2 plasmids can result in quantitatively and qualitatively different humoral immune responses.

Metastasis from human breast cancer cell lines.

Immunodeficient animals, principally nude mice, when used in appropriately designed studies have been shown to be useful for the experimental analysis of human breast cancer metastasis. As with many other human tumors, the implantation of breast cancer cells into an anatomically appropriate tissue (the mammary fatpad) results in increased tumor take and incidence of metastasis for certain cell lines compared with subcutaneous injection. Testing a number of widely available human breast cancer cell lines identified the MDA-MB-435 cell line as the most metastatic, producing lung and lymph node metastases in a high proportion of nude and severe combined immunodeficient (SCID) mice after injection in the mammary fatpad. Mixing human breast cancer cells with normal fibroblasts or with Matrigel also increases the tumor incidence and growth rates in nude mice. Different routes of injection can be used to assess the ability of human breast cancer cells to form metastatic lesions in the lungs (i.v. injection), the liver (injection in the spleen), the brain (direct or intracarotid artery injection) and the bone marrow and bone (injection into the left ventricle of the heart). These different approaches demonstrate the potential of experimental studies of human breast cancer growth and metastasis using immunodeficient mice; this model is valuable for experiments that test the role of metastasis-associated genes and the efficacy of novel forms of therapy.

Experimental regulation of granulomatous reactions around Schistosoma japonicum eggs implanted into the liver of mice

Hirata M., Takushima M., Kaoe M. and Fukuma T. 1991. Experimental regulation of granulomatous reactions around Schistosoma japonicum eggs implanted into the liver of mice. International Journal for Parasitology 21: 801–806. Using a liver model, various granulomatous responses against Schistosoma japonicum eggs were studied in C57BL/6 mice immunized with tissue-extracted eggs prior to challenge implantation with freshly laid eggs. In mice receiving two ip injections of 20,000 eggs, there was little effect on early granuloma formation. Three weeks after implantation, however, tissue reaction accompanied by marked fibrosis was significantly augmented, compared to that in the untreated mice. In contrast, when mice were given four ip injections, the early reaction was accelerated and the subsequent fibrosis came to an end earlier than in the twice immunized or untreated mice. Different routes of injection produced differing effects on 2-week granulomas, with an augmented reaction following two sc injections and a diminished reaction following the same number of ip injections. Histologically, the diminished reaction was characterized by less cellularity, especially in the case of eosinophil infiltration.

Effect of the injection site on the bioavailability of amoxycillin trihydrate in dairy cows

The pharmacokinetic behaviour of amoxycillin sodium and amoxycillin trihydrate‐20% aqueous suspension was studied in a group of five dairy cows. Amoxycillin sodium was administered intravenously and amoxycillin trihydrate‐20% by four different routes of administration: subcutaneously in the dewlap, intramuscularly in the lateral neck, M. triceps, and buttock (M. semitendineus). The dose level for both drug formulations was 3.83 ± 0.47 mg/kg.The mean plasma concentration–time curve(Cp)for intravenous amoxycillin sodium administration could be described mathematically by the biexponential equation Cp= 15.6 e‐0.033t+ 1.04 e‐0.0091t. The areas under the plasma concentration–time curve (AUC's) obtained after the intravenous injections of sodium amoxycillin were used as references for the bioavailability studies of the four routes of amoxycillin trihydrate administration.Intramuscular injections into the lateral neck or into the M. triceps resulted in similar systemic bioavailabilities, being at 12 h post injection (p.i.) 76.2 and 79.2% of the administered dose. The biological half‐lives (t1/2) were similar, being 6.2 and 6.9 h, respectively. After subcutaneous injection into the dewlap or intramuscular injection into the buttock lower bioavailabilities at 12 h p.i. were observed (24.1 and 49.2%, respectively). The plasma amoxycillin concentration was persistently low. The half‐lives of plasma amoxycillin disposition after the buttock and dewlap injections were 13.2 and 44 h, respectively.The plasma concentrations obtained were compared with minimal inhibitory concentrations (MIC) against pathogenic bacteria with respect to the theoretical design of effective antibacterial therapy. The differences observed serve to emphasize the fact that more attention should be paid to the effect of the route of administration on the biological bioavailability of a drug, with particular reference to studies on clinical efficacy. Pre‐slaughter withdrawal times were suggested for the different routes of injection of these two drug formations.

Effect of stimulation on the incorporation of 14C from glial and neuronal specific substrates into brain proteins in vivo and in vitro.

The incorporation of amino acids into brain proteins following brachial plexus stimulation (BPS) was studied in anaesthetised Sprague-Dawley rats following injection of radioactive precursors of both neuronal and glial compartments. Following intraperitoneal injection of [14C]glucose, which is the major neuronal pool precursor, BPS resulted in a significant increase of 37% (P < 0.001) in the incorporation of carbon from [14C]glucose into TCA-insoluble proteins in the contralateral sensorimotor cortex as compared with the ipsilateral area of the same animal. This increase was abolished totally when tetrodotoxin (10 micrograms ml-1) was applied topically to the surface of the stimulated area. Following intraperitoneal injection of [14C]acetate, which is considered to by mainly a glial cell precursor, the same afferent electrical stimuli caused a significant decrease of 21% in the incorporation of amino acids into proteins in the stimulated versus unstimulated sensorimotor cortex. With [4-(3)H]phenylalanine or [1-(14)C]leucine as precursors a significant decrease (12%) or no change was recorded, respectively. A similar decrease in protein synthesis in the stimulated sensorimotor cortex was achieved using different routes of injection. No significant changes were observed in the ratio of the specific radioactivities of the total amino acids of the two hemispheres using either precursor. In vitro, synaptosomes showed a large increase in incorporation into proteins after treatment with electrical pulses, both with [14C]glucose and with [U-14C]acetate as precursors.

Early distribution of radioactive liposomes and scrapie infectivity in mouse tissues following administration by different routes

Mice were injected with 99mtechnetium-labelled liposomes or with a standard homogenate of scrapie-affected brain. Four different routes of injection were used, intracerebral (i.c.), intravenous (i.v.), intraperitoneal (i.p.) and subcutaneous (s.c.). Blood and a range of other tissues were removed between 5 min and several hours after injection. The tissues included spleen and salivary gland in which appreciable agent replication (or at least accumulation) is known to occur and, liver where replication of scrapie agent does not occur. Within 30 min of injection by any of the four routes used, radioactivity, and infectivity were detected in tissues remote from the site of injection. The distribution of radioactivity in various tissues was similar to that of infectious scrapie agent, but in both cases it occurred much more rapidly after i.v. injection than after injection by the i.p. or s.c. route. It is suggested that the 1000-fold reduced efficiency of scrapie infection by the s.c. route, compared to the i.v. route, is related to the extensive localisation of inoculum at the site of injection. Despite the relatively high efficiency of infection of the i.v. route, much of the detectable agent is taken up by liver where the agent does not replicate. It is concluded that only a very small proportion of the agent injected i.v. (and presumably by other routes) is involved in establishing scrapie infection of mice.

Immunological reconstitution of T-cell-deprived mice: I. Inability of thymosin to restore spleen cell mitogen and tumor allograft response

The effect of thymosin fraction-5 therapy on the response of spleen cells to T-dependent mitogens was investigated in adult thymectomized mice (ATx). Although thymosin fraction 5 potentiated thymocyte proliferation in mixed lymphocyte reactions, it failed to restore or maintain spleen cell Con A and PHA responsiveness in ATx mice after therapy using different routes of injection, doses, vehicle, length of treatment, and post-ATx age recipients. Thymosin fraction-3 or -5 therapy also did not restore tumor allograft response in ATx mice depleted of long-lived T-derived lymphocytes by anti-thymocyte serum treatment. By contrast, newborn thymus grafts were completely effective in reconstituting tumor allograft rejection. These results suggest that maturation of the above functional activities of T-derived lymphocytes depends on the thymus microenvironment or that thymosin is deficient in a thymic humoral factor regulating this maturation in the periphery.

Incorporation of label from(1- 14 C)ethanol into the glutamate-glutamine pools of rat brain in vivo.

Glutamate and glutamine were isolated from the brains of rats given [1-(14)C]ethanol by three different routes of injection with and without carrier ethanol or acetate. Without exception, the specific radioactivity of brain glutamine was 3- to 5-fold that of brain glutamate. The route of injection had no consistent effect on the results. These results indicate that small amounts of ethanol are oxidized by rat brain in the compartment responsible for the so-called ;small metabolic pool' of brain glutamate.

Mercaptoethanol-Sensitive Antibody Production in Germ-Free Mice and Guinea Pigs

Summary Germ-free and nongerm-free guinea pigs and mice were injected intraperitoneally or in the footpads with soluble I131 bovine γ-globulin (BγG) or BγG precipitated with alum. The germ-free guinea pigs responded to these injections in a manner similar to the nongerm-free guinea pigs. Germ-free mice injected intraperitoneally also responded in the same manner as nongerm-free mice, but germ-free mice injected in the footpads produced only a delayed 19 S antibody response while nongerm-free mice injected in the footpads demonstrated a prompt antibody response with a 19 S-to-7 S conversion. It is suggested that this difference in antibody response in germ-free mice after different routes of injection indicates that the number of “potentially competent” cells available to a given antigen may determine the type of antibody produced.

THE CUTANEOUS REACTIVITY OF GUINEA PIGS TO PURE. PROTEIN ANTIGENS. II. THE EFFECTS OF DIFFERENT ROUTES OF INJECTION OF ANTIGEN AND ADJUVANT.

THE CUTANEOUS REACTIVITY OF GUINEA PIGS TO PURE. PROTEIN ANTIGENS. II. THE EFFECTS OF DIFFERENT ROUTES OF INJECTION OF ANTIGEN AND ADJUVANT.