Avidin-biotin Peroxidase Complex Technique Research Articles

GLP-1 Localisation and Proglucagon Gene Expression in Healthy and Diabetic Mouse Ileum.

IntroductionGlucagon-like peptide-1 (GLP-1) is a polypeptide that is mainly produced by intestinal L cells and is encoded by the proglucagon gene. In this study, GLP-1 localisation was investigated in the ileum of healthy and diabetic mice by immunohistochemistry and proglucagon gene expression was assayed by reverse transcription-polymerase chain reaction.Material and MethodsThis study included 18 male Balb/c mice that were divided into diabetic, sham, and control groups. Mice in the diabetic group received 100 mg/kg of streptozotocin. Immunohistochemical expression of GLP-1 was determined using the avidin–biotin–peroxidase complex technique, and proglucagon gene expression was determined by RT-PCR.ResultsAnalysis of GLP-1 immunohistochemical localisation showed that GLP-1-immunopositive cells (L cells) were present between epithelial cells in the intestinal crypts. The intensity and localisation of GLP-1 immunoreactivity were similar among the mice in all the groups. Proglucagon gene expression levels were also statistically similar among the mice in all the groups.ConclusionNo difference was demonstrated among the mice in the diabetic, sham, or control groups with respect to proglucagon gene expression and GLP-1 localisation in the ileum, suggesting that diabetes does not affect proglucagon gene expression in the ileum.

Cultural and immunohistochemical evaluation of bacterial agents in caprine pneumonia in Nigeria

Goats are important protein-producing animals but pneumonia, in spite of the huge investments in small ruminant production, remains a major limitation. This study evaluates the prevalence of important bacterial pathogens associated with caprine pneumonia in Nigeria using cultural and immunohistochemical techniques. One hundred and fifty goat lungs were randomly examined from macroscopic, cultural isolation and microscopic changes using standard techniques. The common bacteria identified were formalin inactivated for bacterine and polyclonal antibody production. Immunohistochemical staining was performed against the bacteria antigens using the avidin-biotin peroxidase complex technique. The data were presented in frequencies and percentages, and compared using non-parametric statistics at α = 0.05. The pathology in the caprine lungs included broncho-interstitial pneumonia (41), interstitial pneumonia (27), and bronchopneumonia (72). Ten of the lungs were normal. Bacterial isolation yielded Mannheimia haemolytica (40%), Pasteurella multocida (20%), Escherichia coli (13%), Klebsiella pneumoniae (10%), Streptococcus sp. (8%), Baccillus sp. (5%), and Pseudomonas sp. (3%). Mannheimia haemolytica (Mh) and Pasturella multocida (Pm) were the most common isolated bacteria. The bacterial antigens were detected on the surface of respiratory bronchiolar, and alveolar epithelia, within inflammatory exudate and in cytoplasm of alveolar macrophages. One hundred twenty (80%) of the caprine lungs were positive for the Mh and Pm antigens; 47 for Mh, 59 for Pm, 14 for both, and 30 negative for either antigens. Significant differences were observed in the staining intensities of the antigens. This study validated Mannheimia haemolytica and Pasteurella multocida as most important bacterial pathogens in caprine pneumonia in Nigeria. Thus, adequate control of caprine pneumonia should consider bacterine or multivalent vaccines incorporating these important bacteria to curb morbidity and mortality of goats.

Immunohistochemical localisation and effect of matrix metalloproteinases and their inhibitors on canine spontaneous periodontitis

Periodontitis is commonly observed in dogs. In human medicine, it is well documented that matrix metalloproteinases (MMPs) are involved in the destruction of the periodontium. Therefore, the aim of this...

Resveratrol Exacerbates Both Autoimmune and Viral Models of Multiple Sclerosis

The polyphenol compound resveratrol is reported to have multiple functions, including neuroprotection, and no major adverse effects have been reported. Although the neuroprotective effects have been associated with sirtuin 1 activation by resveratrol, the mechanisms by which resveratrol exerts such functions are a matter of controversy. We examined whether resveratrol can be neuroprotective in two models of multiple sclerosis: experimental autoimmune encephalomyelitis (EAE) and Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD). EAE was induced in C57BL/6 mice, which were fed a control diet or a diet containing resveratrol during either the induction or effector phase or through the whole course of EAE. SJL/J mice were infected with TMEV and fed a control diet or a diet containing resveratrol during the chronic phase of TMEV-IDD. In EAE, all groups of mice treated with resveratrol had more severe clinical signs than the control group. In particular, resveratrol treatment during the induction phase resulted in the most severe EAE, both clinically and histologically. Similarly, in the viral model, the mice treated with resveratrol developed significantly more severe TMEV-IDD than the control group. Thus, surprisingly, the resveratrol treatment significantly exacerbated demyelination and inflammation without neuroprotection in the central nervous system in both models. Our findings indicate that caution should be exercised in potential therapeutic applications of resveratrol in human inflammatory demyelinating diseases, including multiple sclerosis.

Targeting Inflammatory Demyelinating Lesions to Sites of Wallerian Degeneration

In Theiler’s murine encephalomyelitis virus (TMEV) infection, an animal model for multiple sclerosis (MS), axonal injury precedes inflammatory demyelinating lesions, and the distribution of axonal damage present during the early phase of infection corresponds to regions where subsequent demyelination occurs during the chronic phase. We hypothesized that axonal damage recruits inflammatory cells to sites of Wallerian degeneration, leading to demyelination. Three weeks after TMEV infection, axonal degeneration was induced in the posterior funiculus of mice by injecting the toxic lectin Ricinus communis agglutinin (RCA) I into the sciatic nerve. Neuropathology was examined 1 week after lectin injection. Control mice, infected with TMEV but receiving no RCA I, had inflammatory demyelinating lesions in the anterior/lateral funiculi. Other control mice that received RCA I alone did not develop inflammatory lesions. In contrast, RCA I injection into TMEV-infected mice induced lesions in the posterior funiculus in addition to the anterior/lateral funiculi. We found no differences in lymphoproliferative responses or antibody titers against TMEV among the groups. This suggests that axonal degeneration contributes to the recruitment of inflammatory cells into the central nervous system by altering the local microenvironment. In this scenario, lesions develop from the axon (inside) to the myelin (outside) (Inside-Out model).

Immunohistochemical localization of inhibin and activin subunits, activin receptors, and Smads in ovarian clear cell adenocarcinoma

Our purpose was to examine the immunolocalization of inhibin, activin and the activin signaling system in ovarian clear cell adenocarcinoma tissue. Tissue samples of ovarian clear cell adenocarcinoma were collected from ten women. The patients' ages ranged from 41 to 71 years (mean +/- standard deviation; 53.4+/-9.9). Their surgicopathological staging was based on the criteria of the International Federation of Gynecology and Obstetrics (FIGO); there were 5 patients with stage I, 3 with stage II, and 2 with stage III. We examined the immunohistochemical localization of inhibin/activin alpha-subunit, betaA-subunit, activin A, and activin receptor types IA, IB, IIA, IIB, Smad2, Smad3 and Smad4 using an avidin-biotin peroxidase complex technique. With the antibodies against the betaA-subunit, and activin A, we observed positive immunoreactive staining in the cytoplasm of clear cell adenocarcinoma, whereas we did not observe any staining for the alpha-subunit of inhibin. We observed positive immunoreactive staining in the cell cytoplasm and surface with the antibodies against the activin receptors, types IA, IB, IIA and IIB. We observed positive immunoreactive staining in the cytoplasm and nucleus with the antibodies against the Smad2, Smad3 and Smad4. It is suggested that activin A, not inhibins, might be secreted by ovarian clear cell adenocarcinoma, and that an activin signal transduction pathway might exist in ovarian clear cell adenocarcinoma.

Intracellular localization of NS3 and C proteins in chronic hepatitis C

The cellular localization of hepatitis C virus (HCV) proteins in chronic hepatitis C remains a debatable issue. Aim of the studies included cellular and subcellular localization of two HCV proteins, NS3 and C protein, in liver biopsies from 20 children with chronic hepatitis C employing commercially available monoclonal antibodies and a semiquantitative technique of the proteins expression. In the studies advantage was taken of (Strept)avidin-biotin peroxidase complex and ImmunoMax technique. The cytoplasmic localization of both proteins dominated over nuclear localization. The total amount of NS3 protein exceeded that of C protein. At the ultrastructural level, we observed both proteins in endoplasmic reticulum membranes and mitochondria, but small amount of both proteins was seen also in cell nuclei. The amount of either protein did not correlate with liver histology in our patients. No correlation could be disclosed between content of both proteins and HCV RNA levels in serum. Content of NS3 demonstrated negative correlation with activity of alanine transaminase. In conclusion, the new aspect in present studies involved localization of C and NS3 proteins at the levels of light and electron microscopy in children. For the first time also intramitochondrial localization of NS3 protein was demonstrated.

Expression of epidermal growth factor receptor, ERBB2 and KIT in adult soft tissue sarcomas

Little is known about the expression of receptor tyrosine kinases in adult soft tissue sarcomas (STS). In the current study, the authors analyzed the expression of epidermal growth factor receptor (EGFR), ERBB2, and KIT in 281 patients with STS who were treated in a single institution. Verification of the presence of an association with prognosis was performed. The current study included 281 adult patients with STS of the extremity and trunk who were diagnosed and treated in the National Cancer Center, Tokyo. Expression was assessed using immunohistochemical stains for EGFR, ERBB2, and KIT on formalin-fixed, paraffin-embedded tissue sections by standard avidin-biotin peroxidase complex technique and EGFR detection system. Positive staining of EGFR was observed in 168 of 281 (60%) patients. Positive staining was common in pleomorphic malignant fibrous histiocytomas (89%), myxofibrosarcomas (89%), synovial sarcomas (76%), malignant peripheral nerve sheath tumors (89%), and leiomyosarcomas (73%). It was less common in well differentiated liposarcomas (38%), fibrosarcomas (36%), and myxoid liposarcomas (6%). In contrast, positive staining of ERBB2 and KIT was very limited. Increased levels of EGFR were significantly associated with a decreased probability of overall survival (P = 0.01), although by univariate analysis; probability of overall survival at 5 years was 64% in patients with increased levels of EGFR and 79% in patients without such overexpression. The overexpression of EGFR was significantly associated with histologic grade (P < 0.001). Moreover, stratified log-rank test revealed that there is an interrelation between EGFR overexpression and histologic grade. EGFR overexpression was found to be a negative prognostic factor of adult STS, which is strongly associated with histologic grade. STS patients with EGFR overexpression may benefit from treatment with currently available biospecific inhibitors for EGFR.

Bromodeoxyuridine Incorporation in Differentiated Thyroid Cancer

Objective: Bromodeoxyuridine (BUdR), a thymidine analog, is incorporated into the DNA in the synthetic (S) phase of the cell cycle. BUdR uptake quantifies DNA synthesis and helps predict the prognosis of various cancers. We, therefore, studied BUdR uptake in differentiated thyroid cancers to determine whether it correlates with other clinical prognostic factors in patients with differentiated thyroid cancer. Materials and Methods: Fresh thyroid cancer tissue from 45 patients was incubated in 10 μm BUdR in Dulbecco's modified Eagle medium with 10% fetal calf serum for 1 hour at 37℃, fixed in 70% ethanol and embedded in paraffin. Five tm sections were deparaffinized, denatured, and incubated for 1 hour in 1:3000 purified monoclonal antibodies. The immunohistochemical staining was performed using the avidin-biotin peroxidase complex technique. The extent of BUdR incorporation was measured using the labeling index (% of total cells labeled with BUdR). Results: BUdR labeling index was lower in good risk cancers confined to the thyroid (DeGroot Class Ⅰ) or regional nodes (DeGroot Class Ⅱ) (0.65%±0.23%, N=26) than in poor risk cancers with local invasion (DeGroot Class Ⅲ) or distant metastases (DeGroot Class Ⅳ) (1.13%±0.67%, N=19) (P=0.002 using the t-test). BUdR labeling index was similarly lower in cancers from low risk patients with AGES score ＜4 (0.66%±0.27%, N=29) than those from high risk patients with AGES score ≥4 (1.l9%±0.69%, N=16) (P=0.001 using the t-test). BUdR labeling index correlated with patient age (r=0.313, p=0.00l), and tumor size (r=0.479, p=0.00l). Six patients had tumor BUdR labeling indexes greater than 1.5%. All had DeGroot Class III or IV cancers and had AGES scores greater than 4. Conclusions: We concluded that BUdR labeling index correlated with tumor size, patient age, and risk classification of DeGroot Class or AGES score in patients with differentiated thyroid cancer, and it may become a useful factor in predicting thyroid cancer behavior.

Immunohistochemical Recognition of the Epidermal Growth Factor Receptor by the h-R3 Antibody in the Skin of Experimental Animals

The h-R3 is a humanized monoclonal antibody (Mab) that binds to the external domain of the human epidermal growth factor receptor (EGF-R). It has being used for the treatment of head and neck tumors. Since it is known that different animal species have different EGF-R amino acid sequences, it raises the handicap that, a priori, the animal species relevant for the pharmacodynamic, pharmacokinetic, and toxicologic studies of this Mab are unknown. To elucidate relevant laboratory animal species, the authors investigated the immunohistochemical recognition by h-R3 Mab of EGF-R in small skin biopsy samples obtained from NMRI nu/nu, C57Bl/6, and OF-1 mice; Sprague-Dawley rats; Hartley guinea pigs; New Zealand rabbits; and Cercopithecus aethiops and Macaca fascicularis monkeys. Additionally, three human skin biopsies were obtained from trauma victims. The immunolocalization of EGF-R in different tissue sections was performed using the avidin-biotin peroxidase complex (ABC) technique. The skin sections from different tissues were tested with the biotinylated h-R3 Mab or with a biotinylated irrelevant Mab, used as negative control. The staining intensity was qualified as:-, no staining; +, weak staining; ++, moderate staining; +++, strong staining. Macaca fascicularis monkey, New Zealand rabbit, and OF-1 mouse skins had a strong staining intensity; Cercopithecus aethiops monkey and Sprague-Dawley rat skins showed a moderate to strong staining intensity; C57Bl/6 mouse skins showed a moderate staining intensity. No staining was observed in the skins from Hartley guinea pigs and MNRI nu/nu mice. The fact that h-R3 Mab recognizes other animal EGF receptors in addition to the human EGF-R makes it possible to perform relevant pharmacodynamic, pharmacokinetic, and toxicologic studies in some laboratory animals.

Vasoactive intestinal peptide and its relationship to tumor stage in colorectal carcinoma: an immunohistochemical study.

Vasoactive intestinal peptide (VIP) is considered to influence cellular proliferation through its action on adenylate cyclase. This study examined VIP in the tumor-neighboring mucosa (TM) and remote normal mucosa (RM) in patients with colorectal carcinoma, and explored its relationship to tumor stage. Immunohistochemical staining of VIP, using the avidinbiotin peroxidase complex technique, was performed on TM and RM from 55 patients, surgically resected colorectal carcinomas. The VIP immunoreactivity in the lamina propria (LP) of TM and RM was semiquantitatively graded, according to the density of VIP immunoreactive fibrous strands, and correlated with clinical characteristics, pathological findings, and tumor stage. VIP immunoreactivity in the LP of TM and RM was found mainly as fibrous strands, some of which were nerve fibers. A few pericryptal myofibroblasts also showed VIP immunoreactivity. The VIP immunoreactivity in the LP was significantly greater in TM than in RM. The VIP immunoreactivity in the LP of TM was marginally greater in lesions with distant metastasis. The VIP immunoreactivity in the LP of RM was significantly greater in lesions with deeper wall penetration, in those with lymph node metastasis, and in those at more advanced stages. These results suggest a possible trophic role of VIP in the progression of colorectal carcinoma, or enhanced VIP secretion secondary to or in parallel with the progression of carcinoma.

Differential expression of matrix metalloprotease-7 in each component of uterine carcinosarcoma.

The aim of this study was to investigate the expression of matrix metalloprotease-7 (MMP-7) in each component of uterine carcinosarcoma. Surgical specimens of primary tumors of uterine carcinosarcomas were obtained from 13 patients. The carcinomatous component consisted of adenocarcinoma with or without squamous differentiation. The sarcomatous component consisted of spindle cell sarcoma, chondrosarcoma and rhabdomyosarcoma, either alone or in combination. The immunohistochemical expression of MMP-7 protein was examined using the avidin-biotin peroxidase complex technique employing the anti-MMP-7 monoclonal antibody. Expression of MMP-7 protein was detected in 9 (69.2%) of 13 adenocarcinoma components, while no staining was observed in any of the sarcomatous components examined. There was a statistically significant difference of the positive rate for MMP-7 between epithelial components and sarcomatous components of uterine carcinosarcoma (p<0.01). In some cases, MMP-7 was abundantly expressed at the invasive front of adenocarcinomas. It is concluded that MMP-7 protein is differentially expressed between the carcinomatous component and the sarcomatous component of uterine carcinosarcoma. Each component of carcinosarcoma may have its own potential for invasion and metastasis. MMP-7 may contribute to the invasive nature or growth capacity of the carcinomatous component of uterine carcinosarcoma, while it may not have a relation to that of the sarcomatous components.

Synaptic contacts between serotonergic and cholinergic neurons in the rat dorsal raphe nucleus and laterodorsal tegmental nucleus

We examined synaptic connectivity between cholinergic and serotonergic neurons in the dorsal raphe nucleus and the laterodorsal tegmental nucleus of the rat. To this purpose we employed two variations (the combination of pre-embedding immunogold–silver intensification with avidin–biotin-peroxidase complex technique and the combination of avidin–biotin–peroxidase/3,3′-diaminobenzidine/silver–gold intensification with avidin–biotin–peroxidase/3,3′-diaminobenzidine reaction) of a double pre-embedding immunoelectron procedure, using primary antibodies against vesicular acetylcholine transporter and serotonin. At the light-microscopic level, serotonin-like immunoreactive neurons in the dorsal raphe nucleus appeared as reddish black and vesicular acetylcholine transporter-like immunoreactive axon terminals were brown colored using a combination of pre-embedding immunogold-silver technique and avidin–biotin–peroxidase complex technique. Serotonin-like immunoreactive fibers projected to the laterodorsal tegmental nucleus. At the electron microscopy level, with both methods we observed in the dorsal raphe nucleus vesicular acetylcholine transporter-immunopositive axon terminals in synaptic contact with serotonin-like immunoreactive dendrites and, to a lesser degree, with serotonin-like immunoreactive cell bodies. These synapses usually were of the symmetrical type. Occasionally we noted, next to vesicular acetylcholine transporter-immunopositive axon terminals, also immunonegative terminals synapsing with the serotonin-like immunoreactive dendrites. In the laterodorsal tegmental nucleus we found serotonin-like immunoreactive axon terminals and immunonegative terminals forming synapses with vesicular acetylcholine transporter-immunoreactive dendrites. Most synapses formed by the serotonin-like immunopositive terminals were of the asymmetrical type. Our results suggest that serotonergic neurons in the dorsal raphe nucleus and cholinergic neurons in the laterodorsal tegmental nucleus may reciprocally influence each other by means of synaptic connectivity. Such connectivity may serve to regulate pain sensation, or be involved in the regulation of the sleeping–waking cycle.

CD31 Immunoreactivity in Small Round Cell Tumors

CD31 has been shown to be a sensitive and specific marker for endothelial differentiation among epithelioid and spindled-pleomorphic human neoplasms. However, the role of this marker in the evaluation of small round cell tumors has not been evaluated. Formalin-fixed, paraffin-embedded tissue sections from 276 small round cell tumors, including 85 Ewing's sarcoma/primitive neuroectodermal tumors (ES/PNET), 52 rhabdomyosarcomas, 10 extraabdominal polyphenotypic small cell tumors, six desmoplastic small cell tumors, 11 neuroblastomas, 23 Wilms' tumors, 20 retinoblastomas, 13 esthesioneuroblastomas, and 56 small cell malignant lymphomas were stained with CD31 (JC/70A, 1:40), using a modified avidinbiotin-peroxidase complex technique, after citrate buffer microwave epitope retrieval. Among nonlymphoid small round cell tumors, four of 85 ES/PNET were at least focally reactive. No other lesion in this group was positive. In contrast, the majority of well-differentiated (11 of 17), intermediately differentiated (two of three), and lymphoblastic lymphomas (three of three) were positive. Small cleaved lymphomas (three of 13 follicular, one of 13 diffuse) were less often reactive, whereas small noncleaved lesions were negative. Although reactivity for CD31 in ES/PNET is uncommon, the presence of platelet/endothelial cell adhesion molecule in a small cell neoplasm should not in isolation be taken as evidence of hematopoietic origin. These results further define the utility of CD31 in the evaluation of human neoplasms.

Oestrogen and progesterone receptors in feline fibroadenomatous change: an immunohistochemical study

The distribution of oestrogen and progesterone receptors was analysed in 18 cases of feline fibroadenomatous change (FFAC) using commercially available specific monoclonal antibodies and the Avidin–biotin peroxidase complex technique on formalin-fixed, paraffin-embedded tissue. In all cases of FFAC, progesterone receptors were detected either in epithelial cells (mostly suprabasal) or in epithelial and stromal cells. Oestrogen receptors were detected in approximately half of these cases, in suprabasal or luminal epithelial cells exclusively. Myoepithelial cells lacked both oestrogen and progesterone receptors. The techniques used have identified the specific cellular distribution of steroid hormones receptors in this hormone-dependent lesion of the feline mammary gland. The results confirm those of previous biochemical analyses with respect to progesterone receptors and add new data concerning the possible involvement of oestrogen receptors and stromal fibroblasts in the hormonal control and development of the lesion.

Distribution of adrenomedullin-like immunoreactivity in the rat central nervous system by light and electron microscopy

Adrenomedullin is a peptide of marked vasodilator activity first isolated from human pheochromocytoma and subsequently demonstrated in other mammalian tissues. Using a polyclonal antiserum against human adrenomedullin-(22–52) amide and the avidin–biotin peroxidase complex technique, we have demonstrated by light and electron microscopy that adrenomedullin-like immunoreactivity is widely distributed in the rat central nervous system. Western blotting of extracts of different brain regions demonstrated the fully processed peptide as the major form in the cerebellum, whereas a 14-kDa molecular species and a small amount of the 18-kDa propeptide were present in other brain regions. Immunoreactive neurons and processes were found in multipolar neurons and pyramidal cells of layers IV–VI of the cerebral cortex and their apical processes, as well as in a large number of telencephalic, diencephalic, mesencephalic, pontine and medullary nuclei. Cerebellar Purkinje cells and mossy terminal nerve fibers as well as neurons of the cerebellar nuclei were immunostained, as were neurons in area 9 of the anterior horn of the spinal cord. Immunoreactivity was also found in some vascular endothelial cells and surrounding processes that probably originated from perivascular glial cells. Electron microscopy confirmed the light microscopy findings and showed the reaction product in relation to neurofilaments and the external membrane of small mitochondria. Immunoreactive terminal boutons were occasionally seen. The distribution of adrenomedullin-like immunoreactivity in the central nervous system suggests that it has a significant role in neuronal function as well as in the regulation of regional blood flow.

Detection of chromogranin A in human gastric adenocarcinomas using a sensitive immunohistochemical technique.

Neuroendocrine cells are often disclosed in human gastric adenocarcinomas and may be recognised by their immunoreactivity towards chromogranin A. However, in dedifferentiated neuroendocrine tumour cells, the chromogranin A content may be reduced making it difficult to detect with conventional immunohistochemical methods. We therefore used a sensitive signal amplification technique in order to evaluate chromogranin A immunoreactivity and thus neuroendocrine differentiation in 40 gastric adenocarcinomas. Neuroendocrine cells were visualised by means of a monoclonal chromogranin A antibody and the avidin-biotin peroxidase complex technique, without and with addition of tyramide signal amplification. Double immunohistochemistry towards chromogranin A and Ki-67 were used to disclose proliferation in the neoplastic cells. A marked increase in the number of carcinomas containing chromogranin A-immunoreactive neoplastic cells was noted when applying the tyramide signal amplification technique. In addition, the number of immunoreactive cells within each tumour increased, and in some cases almost all the neoplastic cells became immunoreactive. Chromogranin A-immunoreactive tumour cells showing signs of proliferation were found in the majority of these carcinomas. In conclusion, we have disclosed widespread immunoreactivity towards chromogranin A in a proportion of gastric adenocarcinomas when enhancing the signal with tyramide signal amplification. Neuroendocrine differentiation is thus a common finding in gastric carcinomas when using sensitive methods.

Electron microscopic observation of μ-opioid receptor in the rat area postrema☆

A simple preembedding avidin–biotin–peroxidase complex technique was used to study the ultrastructural localization of μ-opioid receptor in the rat area postrema. By using low concentrations of the first antiserum for incubation with a short reaction time to 3,3′-diaminobenzidine, the immunostaining was faint at the light microscopic level. However, at the electron microscopic level, strong immunoreaction was observed. μ-Opioid receptors were found to be localized on the postsynaptic membrane of dendrites, extrasynaptic plasma membrane, and the surface of the small, clear vesicles in axon terminals. Of the total 283 immunopositive profiles observed, 68.2% (193 of 283) were dendrites, 29.3% (83 of 283) were axon terminals, and 2.5% (7 of 283) were myelinated axons. No immunostained neuron bodies were found in the present study; 109 μ-opioid receptor immunoreactive dendrites received synapses (56.5%, 109 of 193) from nonimmunoreactive (84.4%, 92 of 109) or immunoreactive (15.6%, 17 of 109) axon terminals, whereas 84 dendrites (43.5%, 84 of 193) were found without receiving synapses. The present study shows that the μ-opioid receptor in the area postrema plays a role mainly at the synapses.

Localization of Nontypeable Haemophilus influenzae Endotoxin in the Middle and Inner Ear During Experimental Otitis Media

Nontypeable Haemophilus influenzae (NTHi) lipooligosaccharide, the major component of H. influenzae endotoxin, was localized in the middle and inner ear subsequent to the resolution of experimental otitis media induced by this pathogen. A monoclonal antibody specific for the lipooligosaccharide of this strain was used to probe sections of middle and inner ear tissue and visualized by means of the avidin-biotin peroxidase complex technique. Sixteen to seventeen days post inoculation with either viable or formalin-inactivated NTHi, endotoxin could be localized in both the middle and inner ear at a time when the middle ear was culture negative. Our data demonstrate that endotoxin shed by NTHi during otitis media penetrates the inner ear and binds to both tissue components and inflammatory cells in both the middle and inner ear.

Lectin histochemistry of laryngeal squamous cell carcinomas

A panel of five biotinylated lectins was applied to study the presence and distribution of membrane carbohydrate residues in the normal laryngeal epithelium and in laryngeal squamous cell carcinomas (SCCs) of 86 patients with the avidin-biotin peroxidase complex technique. The lectin-binding pattern of well-differentiated SCCs was comparable to that of the spinous cells of the normal laryngeal epithelium. In the less differentiated SCCs, staining of the keratinocyte plasma membrane with lectins was either reduced or absent, indicating a decline in the glycosylation of cell surface glycoconjugates. The lectins applied here could be used in the rapid assessment of less-differentiated areas within a laryngeal SCC, but they cannot be regarded as reliable markers of laryngeal keratinocytes undergoing malignant transformation. (Otolaryngol Head Neck Surg 1998;118:886-91.)