In-depth characterization of a new patient-derived xenograft model for metaplastic breast carcinoma to identify viable biologic targets and patterns of matrix evolution within rare tumor types

Metaplastic breast carcinoma (MBC) is a rare breast cancer subtype with rapid growth, high rates of metastasis, recurrence and drug resistance, and diverse molecular and histological heterogeneity. Patient-derived xenografts (PDXs) provide a translational tool and physiologically relevant system to evaluate tumor biology of rare subtypes. Here, we provide an in-depth comprehensive characterization of a new PDX model for MBC, TU-BcX-4IC. TU-BcX-4IC is a clinically aggressive tumor exhibiting rapid growth in vivo, spontaneous metastases, and elevated levels of cell-free DNA and circulating tumor cell DNA. Relative chemosensitivity of primary cells derived from TU-BcX-4IC was performed using the National Cancer Institute (NCI) oncology drug set, crystal violet staining, and cytotoxic live/dead immunofluorescence stains in adherent and organoid culture conditions. We employed novel spheroid/organoid incubation methods (Pu·MA system) to demonstrate that TU-BcX-4IC is resistant to paclitaxel. An innovative physiologically relevant system using human adipose tissue was used to evaluate presence of cancer stem cell-like populations ex vivo. Tissue decellularization, cryogenic-scanning electron microscopy imaging and rheometry revealed consistent matrix architecture and stiffness were consistent despite serial transplantation. Matrix-associated gene pathways were essentially unchanged with serial passages, as determined by qPCR and RNA sequencing, suggesting utility of decellularized PDXs for in vitro screens. We determined type V collagen to be present throughout all serial passage of TU-BcX-4IC tumor, suggesting it is required for tumor maintenance and is a potential viable target for MBC. In this study we introduce an innovative and translational model system to study cell–matrix interactions in rare cancer types using higher passage PDX tissue.

In-depth characterization of a new patient-derived xenograft model for metaplastic breast carcinoma to identify viable biologic targets and patterns of matrix evolution within rare tumor types.

Metaplastic breast carcinoma (MBC) is a rare breast cancer subtype with rapid growth, high rates of metastasis, recurrence and drug resistance, and diverse molecular and histological heterogeneity. Patient-derived xenografts (PDXs) provide a translational tool and physiologically relevant system to evaluate tumor biology of rare subtypes. Here, we provide an in-depth comprehensive characterization of a new PDX model for MBC, TU-BcX-4IC. TU-BcX-4IC is a clinically aggressive tumor exhibiting rapid growth in vivo, spontaneous metastases, and elevated levels of cell-free DNA and circulating tumor cell DNA. Relative chemosensitivity of primary cells derived from TU-BcX-4IC was performed using the National Cancer Institute (NCI) oncology drug set, crystal violet staining, and cytotoxic live/dead immunofluorescence stains in adherent and organoid culture conditions. We employed novel spheroid/organoid incubation methods (Pu·MA system) to demonstrate that TU-BcX-4IC is resistant to paclitaxel. An innovative physiologically relevant system using human adipose tissue was used to evaluate presence of cancer stem cell-like populations ex vivo. Tissue decellularization, cryogenic-scanning electron microscopy imaging and rheometry revealed consistent matrix architecture and stiffness were consistent despite serial transplantation. Matrix-associated gene pathways were essentially unchanged with serial passages, as determined by qPCR and RNA sequencing, suggesting utility of decellularized PDXs for in vitro screens. We determined type V collagen to be present throughout all serial passage of TU-BcX-4IC tumor, suggesting it is required for tumor maintenance and is a potential viable target for MBC. In this study we introduce an innovative and translational model system to study cell-matrix interactions in rare cancer types using higher passage PDX tissue.

Glossopharyngeal nerve regeneration is essential for the complete recovery of quinine-stimulated oromotor rejection behaviors and central patterns of neuronal activity in the nucleus of the solitary tract in the rat.

The peripheral, central, and behavioral consequences of glossopharyngeal nerve transection (GLX), regeneration, and the prevention of regeneration on the quinine-elicited responses of adult rats were concurrently examined. Oromotor taste reactivity (TR) was videotaped during intraoral infusion of 7 ml of either quinine (3 mm) or distilled water at 17, 52, or 94 d after surgery. We confirmed previous findings by showing that 17 d after neurotomy, (1) the number of circumvallate (CV) and foliate taste buds, (2) gapes (a characteristic aversive TR response), and (3) the number of Fos-like immunoreactive (FLI) neurons in the gustatory NST (gNST), particularly in the medial portion (subfield 5) of the rostral central subdivision (RC), were all severely attenuated in GLX rats. We extended these findings by showing that these lesion-induced effects were enduring when the GL did not regenerate (up to 94 d). In contrast, when the GL regenerated, as few as 52 d were sufficient to re-establish quinine-elicited TR, especially gaping, and FLI expression in RC, particularly within subfield 5, to values comparable with quinine-stimulated sham-operated rats. Evidently, the gNST maintains its potential to restore accurately the organization of neural activity that is disrupted by nerve injury, as assessed by FLI, ultimately leading to the return of normal protective oromotor responses, provided the nerve regenerates. This recovery was complete despite the reappearance of a reduced population of CV taste buds ( approximately 75% control values) and may relate to peripheral and/or central changes that occur in tandem with regeneration of the GL.

Glossopharyngeal nerve transection eliminates quinine-stimulated fos-like immunoreactivity in the nucleus of the solitary tract: implications for a functional topography of gustatory nerve input in rats.

The relationship between specific gustatory nerve activity and central patterns of taste-evoked neuronal activation is poorly understood. To address this issue within the first central synaptic relay in the gustatory system, we examined the distribution of neurons in the nucleus of the solitary tract (NST) activated by the intraoral infusion of quinine using Fos immunohistochemistry in rats with bilateral transection of the chorda tympani (CTX), bilateral transection of the glossopharyngeal nerve (GLX), or combined neurotomy (DBLX). Compared with nonstimulated and water-stimulated controls, quinine evoked significantly more Fos-like-immunoreactive (FLI) neurons across the rostrocaudal extent of the gustatory NST (gNST), especially within its dorsomedial portion (subfield 5). Although the somatosensory aspects of fluid stimulation contributed to the observed increase in FLI neurons, the elevated number and spatial distribution of FLI neurons in response to quinine were remarkably distinguishable from those in response to water. GLX and DBLX produced a dramatic attenuation of quinine-evoked FLI neurons and a shift in their spatial distribution such that their number and pattern were indiscernable from those observed in water-stimulated controls. Although CTX had no effect on the number of quinine-evoked FLI neurons within subfield 5 at intermediate levels of the gNST, it produced intermediate effects elsewhere; yet, the spatial distribution of the quinine-evoked FLI neurons was not altered by CTX. These findings suggest that the GL provides input to all FLI neurons responsive to quinine, however, some degree of convergence with CT input apparently occurs in this subpopulation of neurons. Although the role of these FLI neurons in taste-guided behavioral responses to quinine remains speculative, their possible function in oromotor reflex control is considered.

Antifungal therapy during pregnancy.

Careful consideration of the benefit to the mother and the risk to the fetus is required when prescribing antifungal therapy in pregnancy. Imidazoles are considered safe as topical therapy for fungal skin infections during pregnancy. Nystatin is minimally absorbed and is effective for vaginal therapy. Although vaginal use of the imidazoles is probably safe during the later stages of pregnancy, their systemic absorption is higher than when applied to the skin. The systemic antifungal drug with which there has been the most experience in pregnancy is amphotericin B. There have been no reports of teratogenesis attributed to this agent. There is evidence to suggest that fluconazole exhibits dose-dependent teratogenic effects; however, it appears to be safe at lower doses (150 mg/day). Ketoconazole, flucytosine, and griseofulvin have been shown to be teratogenic and/or embryotoxic in animals. Iodides have been associated with congenital goiter and should not be used during pregnancy.

Pipeline architectures for recursive morphological operations.

Introduces efficient pipeline architectures for the recursive morphological operations. The standard morphological operation is applied directly on the original input image and produces an output image. The order of image scanning in which the operator is applied to the input pixels is irrelevant. However, the intent of the recursive morphological operations is to feed back the output at the current scanning pixel to overwrite its corresponding input pixel to be considered into computation at the following scanning pixels. The resultant output image by recursive morphology inherently depends on the image scanning sequence. Two pipelined implementations of the recursive morphological operations are presented. The design of an application-specific systolic array is first introduced. The systolic array uses 3 n cells to process an nxn image in 6 n-2 cycles. The cell utilization rate is 100%. Second, a parallel program implementing the recursive morphological operations and running on distributed-memory multicomputers is described. Performance of the program can be finely tuned by choosing appropriate partition parameters.

Neuroanatomical alterations in the rat nucleus of the solitary tract following early maternal NaCl deprivation and subsequent NaCl repletion.

Restricting the NaCl content in the rat maternal and preweaning diet results in a significant and specific reduction (60%) of chorda tympani nerve responsiveness to sodium stimuli in the offspring. Repletion of dietary sodium at any time during postnatal development results in a complete and persistent recovery of chorda tympani nerve function. To learn whether the maturation of postsynaptic cells are also affected by the early dietary manipulation, dendritic morphology, neuronal and glial densities and numbers were studied within the area of the nucleus of the solitary tract (NTS). Examination of dendritic morphologies in Golgi-Cox stained neurons revealed that cells with multipolar and fusiform somata in the rostral NTS exhibited longer dendrites following dietary NaCl deprivation during development (deprived rats) than in controls. These changes were generally maintained in rats initially deprived of NaCl and then fed a NaCl-replete diet postweaning ("recovered" rats). In contrast, ovoid neurons were not affected by NaCl deprivation but had increases in the lengths of their dendrites following "recovery." Along with dendritic alterations, the packing density of neurons in the rostral NTS was greater in NaCl-deprived rats than in controls, but was similar to controls following "recovery." Glial packing density also increased following deprivation and remained high in "recovered" rats. These results indicate that activity-dependent events as well as events not related to afferent activity (e.g., hormonal changes) may influence the morphological development of NTS neurons. In addition, significant interactions among primary afferent fibers, central neurons, and glia may direct development within the central gustatory system.

Recurrent nocardiosis in a renal transplant recipient.

As the case presented here illustrates, nocardiosis, like other infections in which cell-mediated immunity plays a large defensive role, can relapse after apparent cure and occasionally at times remote from the original infection. Although relapse in patients with transplants has been cited as a reason for continued prophylaxis, only a few of these cases are adequately documented. This case supports the advice of those authors who give suppressive antibiotic therapy for the duration of immunosuppression in transplant recipients recovering from infections due to Nocardia sp. Alternatively, many transplant centers are routinely using TMP/SMX chemoprophylaxis in all solid organ transplantations to prevent opportunistic infections with Pneumocystis and Listeria sp. Primary prophylaxis has also been associated with a decreased incidence of nocardial infections.

Fatal disseminated Conidiobolus coronatus infection in a renal transplant patient.

A case of fatal disseminated fungal infection due to Conidiobolus coronatus in a patient with a renal transplant is described. This organism, known to cause localized infections in otherwise healthy individuals in the tropics, is now recognized as a cause of fatal infection in immunosuppressed hosts. Histologically, localized infections are characterized by lack of vessel invasion and the presence of an eosinophilic sleeve around fungal elements, called the Splendore-Hoeppli phenomenon. The histologic findings in the present case were more typical of mucormycosis, and the correct diagnosis was established only after the organism was isolated and identified in culture.

Dietary sodium chloride deprivation throughout development selectively influences the terminal field organization of gustatory afferent fibers projecting to the rat nucleus of the solitary tract.

In order to determine whether the developing central gustatory system responds to altered sensory experience, terminal fields of the chorda tympani nerve (CT) within the nucleus of the solitary tract (NTS) in control, NaCl deprived, and rats in which CT taste responses "recovered" from NaCl deprivation were investigated via anterograde transport of HRP. Rats fed a low sodium diet (0.03% NaCl) from the third day of gestation to at least 35 days postnatal exhibited both abnormally distributed and irregularly shaped CT terminal fields. Specifically, the dorsal zone of the field was the smallest in controls whereas it was the largest in deprived rats, occupying more medial and caudal territory within the nucleus. The portion of the field immediately ventral to the dorsalmost zone was characterized by a compact, oval shape in control rats and an irregular, broad configuration in deprived rats. Although it has been observed that deprivation-induced changes in the neurophysiology of the CT are reversible, the central morphological alterations reported here remain abnormal. Restoration of 1.0% NaCl in the diet at 28 days postnatally, for at least 60 days, did not result in normal CT terminal fields. The pattern of the field in rats "recovered" from NaCl deprivation was comparable to that found in deprived rats, and the size of the field was three times that found in control and deprived rats. The terminal fields of another nerve containing gustatory afferents, the lingual-tonsilar branch of the glossopharyngeal nerve (LT-IX), were studied for comparison. Interestingly, the pattern of the LT-IX field was not altered by sodium deprivation. The relative size and topography of the LT-IX fields in deprived rats were similar to controls. Thus, sodium deprivation appears to alter selectively the anatomical organization of the CT. Differences in vulnerability between the CT and LT-IX terminal fields may derive from differences in the responsiveness of these nerves to NaCl, and/or to differences in the timing of early neural events.

Binding of [3H]perhydrohistrionicotoxin and [3H]phencyclidine to the nicotinic receptor-ion channel complex of Torpedo electroplax. Inhibition by histrionicotoxins and derivatives.

Histrionicotoxin, a spiropiperidine alkaloid, and twenty-two analogs inhibited binding of [3H]perhydrohistrionicotoxin [( 3H]H12-HTX) and of [3H]phencyclidine [( 3H]PCP) to sites on the acetylcholine receptor-ion complex of Torpedo electroplax membranes. Structural alterations to the nitrogen (secondary amine) or oxygen (alcohol) functions or to the five carbon and four carbon side chain of histrionicotoxin altered the potency versus [3H]H12-HTX and [3H]PCP binding measured in the presence or absence of a receptor agonist, carbamylcholine. Histrionicotoxin itself was 3-fold more potent versus [3H]PCP binding than versus [3H]H12-HTX binding. N-Methylation or O-acetylation increased this difference, while alterations to the side chains either slightly decreased or markedly increased this difference. Histrionicotoxin was some 3.5-fold more potent versus [3H]H12-HTX binding in the presence of carbamylcholine than in its absence. O-Acetylation increased this selectivity for the carbamylcholine-activated state of the receptor channel complex, while alterations in the side chains either reduced or increased the selectivity. Histrionicotoxin was some 2.2-fold more potent versus [3H]PCP binding in the presence of carbamylcholine than in its absence. N-Methylation of O-acetyl-histrionicotoxin greatly increased this selectivity, while alterations in the side chains either reduced or had no effect on selectivity.

Preoperative lymph node evaluation in prostatic cancer patients who are surgical candidates: the role of lymphangiography and computerized tomography scanning with directed fine needle aspiration.

A total of 53 consecutive patients who were candidates for surgical treatment of prostatic cancer underwent preoperative evaluation of the lymph node status by computerized tomography scanning and/or lymphangiography combined with skinny needle aspiration biopsy of any abnormal lymph nodes. In 7 of 14 patients (50 per cent) ultimately found to have stage D1 disease lymphatic metastases were confirmed histologically with needle biopsy alone, thus, obviating the need for pelvic lymph node dissection. Over-all sensitivity, specificity and accuracy rates were 50, 100 and 91.4 per cent, respectively, for computerized tomography scanning with biopsy and 53.8, 100 and 84.1 per cent, respectively, for lymphangiography with biopsy. Computerized tomography scanning and lymphangiography with aspiration biopsy are cost-effective means to identify approximately 50 per cent of the patients who ultimately have lymphatic metastases.

A comparison of clinical course with blood group antigen testing by specific red cell adherence and immunoperoxidase in ureteral and renal pelvic tumors.

Specimens of transitional cell carcinoma of the ureter and renal pelvis from 20 patients were tested for blood group antigenicity using immunoperoxidase and specific red cell adherence methods. The results of antigen testing were correlated with tumor stage and grade as well as the subsequent clinical course of the patients. The specific red cell adherence test was negative in 80 per cent and the immunoperoxidase test was negative in 40 per cent of all tumors. Of the 4 patients with positive specific red cell adherence tests 3 had high grade (II to III), invasive tumors as did 7 of 12 with tumors that were positive by immunoperoxidase testing. Blood group antigen testing did not prove helpful in predicting the clinical course of our patients. In addition, a careful review of previously published data does not support the conclusion that blood group antigen testing is a valuable predictor of upper tract tumor aggressiveness.

Immunohistochemical demonstration of blood group antigens in neoplastic and normal human urothelium: a comparison with standard red cell adherence.

The specific red cell adherence test as a method to detect blood group antigen deletion in urothelial malignancy has been reported to yield approximately 40 per cent false negative results in 0 blood group patients. Our study of multiple sections of 8 normal ureters from blood group 0 patients and more than 220 specimens of transitional cell cancer taken from 48 patients reveals that the immunoperoxidase technique is more specific than the specific red cell adherence method in predicting subsequent invasion in blood group O(H) patients presenting with superficial transitional cell carcinomas (71 compared to 29 per cent) but is no more specific for tumors containing A or B antigens. However, immunoperoxidase staining does improve discernment of underlying histologic detail and, thereby, facilitates recognition of false positive antigen testing associated with squamous and adenomatous metaplasia. Areas of squamous and adenomatous metaplasia in specimens we tested were frequently antigen positive in invasive tumors. Therefore, we believe that these areas must be disregarded in determining antigen deletion in transitional cell carcinomas.

Benzomorphan interactions with acetylcholine receptor complexes from Torpedo.

Benzomorphan interactions with acetylcholine receptor complexes from Torpedo californica electric organ were investigated using radiolabelled probes. All of the benzomorphans had minimal affinity for the acetylcholine binding site. Four benzomorphans (N-allylnormetazocine, cyclazocine, phenazocine and pentazocine) inhibited the binding of tritiated perhydrohistrionicotoxin and phencyclidine to ion channel sites with IC50 values between 0.4 and 5 microM. Two other benzomorphans, ketazocine and ethylketocyclazocine, were much less active. Thus, ion channel affinity is not limited to benzomorphans which stimulate sigma-opiate receptors, and is not a certain indication of psychotomimetic potency.

A new sequence-specific endonuclease from Streptococcus faecalis subsp. zymogenes.

A new sequence-specific endonuclease, SfaI, has been partially purified from Streptococcus faecalis subsp. zymogenes. SfaI recognizes the tetranucleotide sequence 5'G-G-C-C 3' 3' C-C-G-G 5' and cleaves it at the sites indicated by the arrows.

Enhancement of chlorcyclizine teratogenicity in the rat by coadministration of calcium chelating agents.

Chlorcyclizine and structurally related drugs induce a high incidence of cleft palate and skeletal malformations in fetal rats. We have shown previously that these teratogens bind tightly and reversibly to chondroitin sulfate of cartilage and compete with calcium for binding. Experiments reported here demonstrate that co-administration of calcium chelating agents with chlorcyclizine significantly increases both the frequency of malformations and retention of [14C] chlorcyclizine by embryos. Retention of radioactive teratogen by embryos is inverse to retention of [45Ca]calcium. These findings suggest that drug binding to embryonic glycosaminoglycans is involved in the pathogenesis of malformations produced by chlorcyclizine.