Hierarchical Corannulene-Based Materials: Energy Transfer and Solid-State Photophysics.

We report the first example of a donor-acceptor corannulene-containing hybrid material with rapid ligand-to-ligand energy transfer (ET). Additionally, we provide the first time-resolved photoluminescence (PL) data for any corannulene-based compounds in the solid state. Comprehensive analysis of PL data in combination with theoretical calculations of donor-acceptor exciton coupling was employed to estimate ET rate and efficiency in the prepared material. The ligand-to-ligand ET rate calculated using two models is comparable with that observed in fullerene-containing materials, which are generally considered for molecular electronics development. Thus, the presented studies not only demonstrate the possibility of merging the intrinsic properties of π-bowls, specifically corannulene derivatives, with the versatility of crystalline hybrid scaffolds, but could also foreshadow the engineering of a novel class of hierarchical corannulene-based hybrid materials for optoelectronic devices.

Photophysics of GFP-related chromophores imposed by a scaffold design.

In this paper, a rigid scaffold imposes the photophysics of chromophores with a benzylidene imidazolidinone core by mimicking the ß-barrel structure of the green fluorescent protein (GFP) and its analogs. The designed artificial frameworks maintain fluorescence responses and, therefore, conformational rigidity of typically non-emissive GFP-related chromophores. To replicate a small weight percent of the chromophore inside the natural GFP, two synthetic approaches were utilized: coordinative immobilization and non-coordinative inclusion. Despite low chromophore loading in the rigid matrix, both approaches resulted in formation of photoluminescent hybrid materials. Furthermore, the rigid scaffold dictates chromophore fluorescence by replicating its behavior in solution or the solid state. The presented results open an avenue for utilization of rigid scaffolds in the engineering of materials with tunable photoluminescence profiles for a variety of practical applications.

Redox-Active Corannulene Buckybowls in a Crystalline Hybrid Scaffold.

A porous crystalline corannulene-containing scaffold, which combines the periodicity, dimensionality, and structural modularity of hybrid frameworks with the intrinsic properties of redox-active π-bowls, has been prepared. Single-crystal and powder X-ray diffraction, ab initio density functional theory computations, gas sorption analysis, fluorescence spectroscopy, and cyclic voltammetry were employed to study the properties of the novel corannulene derivatives and the buckybowl-based hybrid materials. X-ray diffraction studies revealed the preservation of the corannulene bowl inside the prepared rigid matrix, which offers the unique opportunity to extend the scaffold dimensionality through the buckybowl curvature. Merging the inherent properties of hybrid frameworks with the intrinsic properties of π-bowls opens a new avenue for preparing redox-active materials and potentially improving charge transport in the scaffold.

Microcrystals with enhanced emission prepared from hydrophobic analogues of the green fluorescent protein chromophore via reprecipitation.

Certain synthetic analogues of the green fluorescent protein (GFP) chromophore are almost nonfluorescent in dilute solutions but are strongly light-emissive in the solid state, thus exhibiting aggregation-induced emission (AIE) behavior. In the present work, two such hydrophobic derivatives of the GFP chromophore known to be fluorescent in the crystalline state (p-hexyloxy- and p-dodecyloxybenzylideneimidazolinone) were used to prepare aqueous suspensions of particles via a mild solvent-exchange reprecipitation (RP) method. This evolution was monitored at various experimental conditions by UV-vis absorption and fluorescence spectroscopy, fluorescence microscopy, as well as electron transmission and scanning microscopy. Both compounds spontaneously produced platelet-like microcrystals, the size and shape of which were influenced by the experimental conditions. The dodecyl derivative also led to the concomitant formation of nanofibers, a tendency reinforced by addition of poly(acrylic acid) to the RP medium. The photoluminescence properties of the solids produced by RP were compared to pristine microcrystalline powders obtained by crystallization in an organic solvent. Significant differences in the emission properties were found and are discussed. This study illustrates the fact that AIE fluorescence is strongly dependent on the nature of the particles and hence on the preparation methods. Being aware of these variations is important for the preparation and subsequent use of AIE-active compounds as fluorescent materials.

Ectopic matrix metalloproteinase-9 expression in human brain tumor cells enhances oncolytic HSV vector infection.

Oncolytic herpes simplex virus (oHSV) vectors have shown promise in the treatment of patients with recurrent brain tumors although few complete responses have accrued. Impediments to effective therapy include limited vector distribution on delivery, a consequence of injected virion particle trapping in the tumor extracellular matrix (ECM). To enhance virus delivery and spread, we investigated the use of the matrix metalloproteinase-9 (MMP-9) as a means to degrade collagen type IV, a major component of the ECM and basement membranes of gliomas that is absent in normal brain tissue. SK-N-AS neuroblastoma cells were transduced for constitutive, elevated expression of MMP-9, which did not enhance tumor cell migration in vitro or tumor progression in a murine xenograft brain tumor model. MMP-9 expression improved the distribution and infection of oHSV vectors in spheroid model in vitro. Furthermore, MMP9 induced a vector infection over larger areas of brain tumors in vivo. These results suggest that vector delivery and distribution in vivo can be improved by compromising the ECM, potentially enhancing oncolytic efficacy.

Cytokine gene therapy of gliomas: effective induction of therapeutic immunity to intracranial tumors by peripheral immunization with interleukin-4 transduced glioma cells.

To provide a means for comparing strategies for cytokine gene therapy against intracranial (i.c.) tumors, we generated rat gliosarcoma 9L cells transfected with interleukin-4 (9L-IL4), interleukin-12 (9L-IL12), granulocyte-macrophage colony-stimulating factor (9L-GMCSF) or interferon-alpha (9L-IFNalpha). To simulate direct and highly efficient cytokine gene delivery, cytokine transfected 9L tumors were implanted i.c. into syngeneic rats. i.c. injection led to tumor-outgrowth in the brain and killed most animals, whereas these cell lines were rejected following intradermal (i.d.) injection. Cytokine-expressing i.c. 9L tumors, however, had a greater degree of infiltration by immune cells compared with control, mock-transfected 9L-neo, but to a lesser degree than i.d. cytokine-expressing tumors. Tumor angiogenesis was suppressed in cytokine-transfected tumors. In a prophylaxis model, i.d. vaccination with 9L-IL4 resulted in long-term survival of 90% of rats challenged i.c. with parental 9L; whereas 40% of 9L-GM-CSF, 40% of 9L-IFNalpha and 0% of 9L-IL12-immunized rats were protected. In a therapy model (day 3 i.c. 9L tumors), only i.d. immunization with 9L-IL4 had long-term therapeutic benefits as 43% of rats survived >100 days. These data indicate that peripheral immunization with 9L-IL4 had the most potent therapeutic benefit among various cytokines and approaches tested against established, i.c. 9L tumors.

Immunization with an antigen identified by cytokine tumor vaccine-assisted SEREX (CAS) suppressed growth of the rat 9L glioma in vivo.

We have reported previously that s.c. immunization of rats with IL-4 transduced 9L gliosarcoma cells (9L-IL-4) induced a potent antitumor immunity against intracranial, parental 9L tumors. Subcutaneous implantation of 9L-IL-4 influenced the systemic humoral response, which was demonstrated by Th2-type isotype-switching and the induction of cellular immune responses, which played a critical role in the rejection of tumors. Serological analyses of recombinant cDNA expression libraries (SEREX), has recently emerged as a powerful method for serological identification of tumor-associated antigens (TAAs) and/or tumor rejection antigens (TRAs). Because IL-4 is known to activate B cells and to promote humoral responses, and inasmuch as induction of humoral responses by central nervous system tumors has been reported to be minimal, we investigated whether the induction of a potent humoral immune response against 9L TAAs or TRAs in rats immunized s.c. with 9L-IL4 could be demonstrated. Screening of 5 x 10(5) independent clones of 9L-expression cDNA library for the presence of reactive antibodies in the serum from a 91-IL-4 immunized rat led to the identification of three different TAAs. One 9L TAA (clone 29) was demonstrated to be calcyclin, a member of the S-100 family of calcium-binding proteins. The second 9L TAA (clone 37) was demonstrated to be the rat homologue of the J6B7 mouse immunomodulatory molecule. The third TAA (clones 158 and 171) was determined to be the rat homologue of the mouse Id-associated protein 1 (MIDA1), a DNA-binding, protein-associated protein. Northern blotting demonstrated that message for calcyclin was overexpressed in 9L cells. Message encoding MIDA1 was highly expressed in parental 9L cells and thymus and, to a lesser degree, in testis, suggesting that MIDA1 was comparable with the cancer/testis category of TAAs. Sera obtained from animals bearing 9L-IL-4 were found to have a higher a frequency and titer of antibodies to these antigens when compared with sera obtained from rats bearing sham-transduced 9L (9L-neo) cells. To determine whether immunization with these TAAs induced antitumor immunity, animals were immunized by intradermal injection with expression plasmids encoding calcyclin or MIDA1. Subsequent challenge of rats with parental 9L resulted in significant suppression of tumor growth in animals immunized with MIDA1, but not with calcyclin. These results indicate that MIDA1 is an effective 9L TRA and will be useful for the investigation of specific antitumor immunity in this glioma model. Furthermore, these results suggest that this approach, termed "cytokine-assisted SEREX (CAS)," may serve as an effective strategy for identification of TRAs for in animal-glioma models of cytokine gene therapy, and potentially in humans undergoing cytokine gene therapy protocols as well.

Monoclonal antibodies to vascular endothelial growth factor (VEGF) and the VEGF receptor, FLT-1, inhibit the growth of C6 glioma in a mouse xenograft.

Monoclonal antibodies raised to peptide sequences of vascular endothelial growth factor (VEGF) and the VEGF receptor, FLT-1, inhibited the growth of C6 tumors growing subcutaneously in nude mice. Immunohistochemical analysis demonstrated antibody targeting of blood vessels, tumor cells, and macrophages. A control antibody demonstrated no growth inhibition or tumor uptake. An antibody to FLT- I impaired microvascular maturation and diminished the accumulation of tumor infiltrating macrophages. The antibodies demonstrated affinity for microvasculature and tumor cells in immunohistochemistry of human glioblastoma multiforme. Targeting VEGF and its receptors has potential in the treatment of tumors of the central nervous system. FLT-1 presents an attractive target due to its presence on multiple cell types.

Combination of stereotactic radiosurgery and cytokine gene-transduced tumor cell vaccination: a new strategy against metastatic brain tumors.

OBJECT: To determine if the combination of radiosurgery and tumor cell vaccine would enhance the therapy of metastatic lesions of the central nervous system (CNS), the authors examined the antitumoral effects of radiosurgery and cytokine-transduced tumor cell vaccine. METHODS: Fifty-five rats underwent intracranial implantation of 5 x 10(3) MADB 106 cells. On Day 3 after tumor implantation, 34 rats were inoculated in the flank with nonirradiated MADB 106 cells that had been retrovirally transduced to express granulocyte-macrophage colony-stimulating factor or interleukin-4. Twenty-seven rats (17 animals that had received the vaccine and 10 that had not) underwent radiosurgery performed using a gamma knife at maximum doses of 32 Gy on Day 5. No animals in the untreated group or in the vaccine-alone groups survived longer than 21 days. Animals treated by ra diosurgery alone displayed prolonged survival in comparison with untreated animals (p < 0.0001), but only one of 10 animals survived longer than 55 days. In contrast, 14 of 17 animals that received the combination therapy of radiosurgery and vaccination survived longer than 55 days (p = 0.0003 compared with animals that underwent radiosurgery alone). On Day 55, the long-term survivors were challenged by parental MADB 106 cells, which were implanted in the contralateral hemisphere. All animals from the combination therapy groups survived longer than 50 days after this challenge, but the single survivor from the radiosurgery-alone group died of tumor growth in 27 days. CONCLUSIONS: The combination of radiosurgery and cytokine gene-transduced tumor cell vaccine markedly prolonged animal survival and protected animals from a subsequent challenge by parental tumor cells placed in the CNS. The data provided by this study indicate that this combination therapy represents a strategy that may have clinical applicability for single and/or multiple metastatic brain tumors.

Subnecrotic stereotactic radiosurgery controlling epilepsy produced by kainic acid injection in rats.

OBJECT: Any analysis of the potential role of stereotactic radiosurgery for epilepsy requires the experimental study of its potential antiepileptogenic, behavioral, and histological effects. The authors hypothesized that radiosurgery performed using subnecrotic tissue doses would reduce or abolish epilepsy without causing demonstrable behavioral side effects. The kainic acid model in rats was chosen to test this hypothesis. METHODS: Chronic epilepsy was successfully created by stereotactic injection of kainic acid (8 microg) into the rat hippocampus. Epileptic rats were divided into three groups: high-dose radiosurgery (60 Gy, 16 animals), low-dose (30 Gy, 15 animals), and controls. After chronic epilepsy was confirmed by observation of the seizure pattern and by using electroencephalography (EEG), radiosurgery was performed on Day 10 postinjection. Serial seizure and behavior observation was supplemented by weekly EEG sessions performed for the next 11 weeks. To detect behavioral deficits, the Morris water maze test was performed during Week 12 to study spatial learning and memory. Tasks involved a hidden platform, a visible platform, and a probe trial. After radiosurgery, the incidence of observed and EEG-defined seizures was markedly reduced in rats from either radiosurgically treated group. A significant reduction was noted after high-dose (60 Gy) radiosurgery in Weeks 5 to 9 (p < 0.003). After low-dose (30 Gy) radiosurgery, a significant reduction was found after 7 to 9 weeks (p < 0.04). During the task involving the hidden platform, kainic acid-injected rats displayed significantly prolonged latencies compared with those of control animals (p < 0.05). Hippocampal radiosurgery did not worsen this performance. The probe trial showed that kainic acid-injected rats that did not undergo radiosurgery spent significantly less time than control rats in the target quadrant (p = 0.03). Rats that had undergone radiosurgery displayed no difference compared with control rats and demonstrated better performance than rats that received kainic acid alone (p = 0.04). Radiosurgery caused no adverse histological effects. CONCLUSIONS: In a rat model, radiosurgery performed with subnecrotic tissue doses controlled epilepsy without causing subsequent behavioral impairment.

Effective treatment of experimental glioblastoma by HSV vector-mediated TNF alpha and HSV-tk gene transfer in combination with radiosurgery and ganciclovir administration.

Experiments were carried out in a nude mouse model of human glioblastoma to determine whether gamma-knife radiosurgery combined with herpes simplex virus thymidine kinase (tk) suicide gene therapy and tumor necrosis factor alpha (TNFalpha) gene transfer provided an improved multimodality treatment of this disease. Animals were inoculated intracerebrally with 2 x 10(5) U-87MG human glioblastoma cells to establish brain tumors. At 3 days postinoculation, the tumor region was injected with 2 x 10(6) infectious particles of highly defective herpes simplex viral vectors expressing the viral tk gene with the kinetics of a viral immediate early gene either alone (T.1) or together with TNF alpha (TH:TNF). Subgroups of animals were given daily intraperitoneal injections of ganciclovir (GCV) for 10 days and/or subjected to gamma-knife radiosurgery on the fifth day post tumor-cell implantation. Comparisons of animal survival showed that the TH:TNF vector in combination with radiosurgery and GCV administration provided the most effective therapy; eight of nine animals survived for 75 days compared to four of eight using the next best protocol. These findings suggest that gene therapy in combination with more conventional therapeutic methods may provide an improved strategy for extending the life expectancy of patients afflicted with this ultimately fatal disease.

Histological effects of trigeminal nerve radiosurgery in a primate model: implications for trigeminal neuralgia radiosurgery.

OBJECTIVE: Stereotactic radiosurgical treatment of the proximal trigeminal nerve is used to relieve the pain of trigeminal neuralgia. The mechanism of the radiosurgical effect is not understood. METHODS: Two adult baboons underwent stereotactic magnetic resonance imaging-guided radiosurgery, using a gamma knife. A single 4-mm isocenter was targeted to each proximal trigeminal nerve, just anterior to the pons, to deliver a maximal dose of 80 or 100 Gy (total of four nerves). A nonirradiated baboon brain and nerves served as control specimens. Six months after treatment, magnetic resonance imaging was again performed and the brains and nerves were studied using light and electron microscopy. RESULTS: Magnetic resonance imaging indicated a 4-mm-diameter area of contrast enhancement at the target site in each nerve. All irradiated nerves exhibited axonal degeneration and mild edema at the target, with remnants of some myelinated axons. Large and small myelinated and unmyelinated fibers were affected. No inflammation was observed. Nerve necrosis was identified after 100-Gy treatment. The trigeminal ganglion appeared normal. CONCLUSION: Radiosurgery at 80 Gy causes focal axonal degeneration of the trigeminal nerve. At higher doses, partial nerve necrosis is observed. We think that these effects influence the physiological features of trigeminal neuralgia.

Effects of stereotactic radiosurgery on an animal model of hippocampal epilepsy.

OBJECTIVE: Stereotactic radiosurgery has been shown in small clinical series to reduce or abolish seizures in patients with lesion-related or idiopathic epilepsy. The radiation dose necessary to eliminate epileptogenesis is unknown, and the histological and metabolic effects of radiosurgery remain undefined. We hypothesized that in a rat model of kainic acid-induced hippocampal epilepsy, radiosurgery could provide a significant reduction in seizure frequency while limiting biochemical and structural histological damage to the brain. METHODS: Kainic acid (8 g) was injected into the rat hippocampus using stereotactic targeting. Focal seizures so generated were identified with scalp and depth electroencephalography (EEG). Epileptic rats were randomized to a control group (n = 20) and to radiosurgery groups in which maximum doses of 20, 40, 60, or 100 Gy (8-9 animals per group) were administered. Over a 42-day period, seizure frequency was determined by direct observation for 8 hours per week. Scalp EEG was performed weekly in all animals. Magnetic resonance imaging (MRI) studies (T1- and T2-weighted water-proton and quantitative sodium images) were obtained on Days 7, 21, and 42. RESULTS: As compared with the control group, treated animals showed significant reductions in the number of seizures during each successive week after 20-Gy radiosurgery (P = 0.01-0.002). When we combined the number of seizures observed in the latter half of the study (Weeks 4-6), we found a significant reduction in seizures after 20-Gy (P = 0.007), 40-Gy (P = 0.03), 60-Gy (P = 0.03), and 100-Gy (P = 0.03) radiosurgery as compared with control animals. Increasing doses of radiosurgery correlated with higher percentages of rats that became seizure-free by EEG criteria. MRI-determined total sodium concentration in the injected hippocampus was 49.8+/-3 mmol/L, compared with 42.8 mmol/L on the contralateral side (within normal limits). This significant increase in sodium concentration was present in control rats (because of the kainic acid) and did not change with increasing radiosurgery dose. No parenchymal effects from radiosurgery were identified after 20, 40, and 60 Gy, and only two rats had necrosis at 100 Gy. All animals showed hippocampal injury from kainic acid by proton MRI and histological examination. CONCLUSION: In this rat hippocampal epilepsy model, stereotactic radiosurgery was followed by a significant dose-dependent reduction in the frequency of observed and EEG-defined seizures. These effects were not accompanied by increased radiation-induced structural or metabolic brain injury as assessed by proton and sodium MRI or histological examination. The role of radiosurgery as a new, nondestructive surgical therapy for idiopathic epilepsy warrants further investigation.

Effective cytokine gene therapy against an intracranial glioma using a retrovirally transduced IL-4 plus HSVtk tumor vaccine.

To explore the potential for molecular immunotherapies in the treatment of malignant gliomas, we evaluated the efficacy of subcutaneous tumor cell vaccines in the treatment of intracranial 9L tumors, using 9L gliosarcoma cell lines stably transduced with the murine interleukin-4 cDNA (9L-IL4), the herpes simplex virus-thymidine kinase cDNA (9L-Tk) or both (9L-IL4-Tk). The expression of multiple genes from a single transcript was achieved by incorporating internal ribosomal entry site (IRES) cassettes in the retroviral constructs. Subcutaneous immunization of rats with nonirradiated 9L-IL4 cells or 9L-IL4-Tk cells followed by treatment with ganciclovir (GCV) completely protected the animals from a subsequent intracranial challenge with wild-type 9L cells. In contrast, only 50% of animals immunized with 9L-Tk cells and 0% of 9L-neo immunized animals rejected the same challenge with wild-type 9L. More importantly, treatment of established (day 3) intracranial 9L tumors with genetically engineered tumor cells resulted in long-term survival (> 100 days) for 25-43% of 9L-IL4-Tk immunized animals and for 27% of nonirradiated 9L-IL4 immunized animals. In striking contrast, no 9L-Tk, 9L-neo or irradiated 9L-IL4 immunized animals survived for more than 33 days. As a marker of a cellular immune response, splenocytes from nonirradiated 9L-IL4, 9L-Tk or 9L-IL4-Tk immunized animals produced interferon-gamma (IFN-gamma) in greater amounts than those from 9L-neo immunized or Hank's balanced salts solution (HBSS) treated animals when stimulated with wild-type 9L in vitro. Our findings support the use of tumor cell vaccines expressing the IL-4 and HSVtk genes for the treatment of malignant gliomas.

Bone marrow-derived dendritic cells pulsed with a tumor-specific peptide elicit effective anti-tumor immunity against intracranial neoplasms.

Although the central nervous system (CNS) is often regarded as an immunologically privileged site, it is well established that specific CNS immunoreactivity can be generated through peripheral vaccination with CNS antigens. Dendritic cells (DC) are potent antigen presenting cells of hematopoietic origin that have emerged as a promising tool for cancer immunotherapy capable of evoking significant anti-tumor immunity when pulsed with tumor-associated peptides. To explore a role for DC-based immunization strategies for the treatment of CNS tumors, we developed a brain tumor model using the C3 sarcoma cell line which expresses the tumor-specific, major histocompatibility complex (MHC) class I-restricted peptide epitope E7(49-57). Syngeneic C57Bl/6 mice receiving intravenous (i.v.) injections of bone marrow-derived DCs pulsed with E7 peptide were effectively protected against a subsequent intracerebral challenge with C3 tumor cells. More importantly, this systemic immunization strategy was effective in a therapy model as 67% of animals (10 of 15) with established (day 7) intracerebral C3 tumors treated with 3 weekly injections of E7 peptide-pulsed DCs achieved a long-term survival (>90 days) while no control animals survived beyond day 41. In vivo depletion of CD8+ cells, but not CD4+ or asialo-GM1+ cells, abrogated the efficacy of E7 peptide-pulsed DC therapy of established tumors, indicating a pivotal role of specific CD8+ T-cell responses in mediating the anti-tumor effect. Our findings support the hypothesis that effective CNS anti-tumor immunoreactivity can be generated with DC-based tumor vaccines.

Interlaboratory testing of the transferability of a candidate reference method for total cholesterol in serum.

In 1975 the Centers for Disease Control, in cooperation with the American Association for Clinical Chemistry Cholesterol Reference Method Study Group, began an investigation to develop a reference method for total cholesterol. Five potential reference methods were compared with the definitive method developed by the National Bureau of Standards before the chemical method of Abell et al. (J Biol Chem 1952;195:357-66) was selected as the recommended reference method. Because acceptance of a proposed reference method depends so greatly on the method's capability for transfer to other laboratories by written specifications and instructions, a transferability testing study was designed and conducted with 14 laboratories. The study consisted of preliminary testing of readiness of equipment, reagents, and personnel followed by transferability testing with eight runs on 10 serum pools. Laboratoires that did not meet readiness specifications had higher CVs in the transferability testing. The study demonstrated that the proposed method permits laboratories to attain a CV of less than 1.5% for one laboratory and of less than 3.0% among laboratories. The mean percent bias value was less than 1.0% for six of the 14 laboratories, less than 1.5% for 12, and less than 3.0% for all 14 laboratories.

Effect of tendon pressure on alpha motoneuron excitability.

The purpose of this study was to test the effect of tendon pressure on muscle activity by evaluating changes in the excitability of the motoneurons supplying the muscle. The excitability changes of the motoneurons were assessed by measuring changes in H-reflex amplitudes before, during, and after tendon-pressure application. Twenty-eight of 32 subjects with no known neurological deficit displayed an initial decrease in H-reflex amplitude during tendon pressure (mean = 60% of control values) but returned to 85 percent of control values within the first 5 seconds of pressure and reached 93 percent of control values within 30 seconds of application. The ANOVA revealed significant differences (p less than .05) and the post hoc t test showed these differences to be between control values and those obtained during the first 10 seconds of pressure. These findings support the clinical impression of reduced muscle tone resulting from tendon pressure. If these results are applied to a patient group, the transient behavior of the tendon-pressure response would limit the applicability of the technique to situations demanding an immediate, short-term reduction in muscle activity.

Comparison of methods for obtaining milk samples from well-nourished and malnourished rats.

Milk samples from well-nourished and malnourished rats were obtained at day 14 of lactation by a standard pumping procedure and an alternative procedure in which the pups were allowed to nurse and milk was collected from their stomachs. Samples obtained by the 2 methods from well-nourished rats did not differ in their concentrations of water, protein, and fat. However, among malnourished rats, pumped samples had significantly higher protein and fat concentrations than nursed samples because they were excessively dehydrated. For both dietary groups, a consistently larger proportion of the available milk was obtained by the nursing method. The results suggest that the nursing method is superior to pumping for obtaining milk samples when rats varying in nutritional status are to be compared.