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Intralaboratory validation of alternative endpoints in the murine local lymph node assay for the identification of contact allergic potential: primary ear skin irritation and ear-draining lymph node hyperplasia induced by topical chemicals.

We validated a two-tiered murine local lymph node assay (LLNA) with a panel of standard contact (photo)allergens and (photo)irritants with the aim of improving the discrimination between contact (photo) allergenic potential and true skin (photo)irritation potential. We determined ear weights to correlate chemical-induced skin irritation with the ear-draining lymph node (LN) activation potential. During tier I LLNAs, a wide range of concentrations were applied on three consecutive days to the dorsum of both ears. Mice were exposed to UVA light immediately after topical application to determine the photoreactive potential of some test chemicals. Mice were killed 24 h after the last application to determine ear and LN weights and LN cell counts. It was possible to classify the tested chemicals into three groups according to their threshold concentrations for LN activation and skin irritation: (1) chemicals with a low LN activation potential and no or very low skin irritation potential; (2) chemicals with a marked LN activation potential higher than a distinct skin irritation potential; and (3) chemicals with LN activation potential equal to or lower than their skin irritation potential. Group 1 consisted only of contact allergens, indicating that LN activation in the absence of skin irritation points to a contact allergenic activity. Since groups 2 and 3 comprised irritants and contact allergens, a tier II LLNA protocol was used to finally differentiate between true irritants and contact allergens. Briefly, mice were pretreated with mildly to moderately irritating concentrations of the chemical to the shaved back and after 12 days were challenged on the ears as described above in order to elicit a contact allergenic response in the ear skin and the ear-draining LN. With this approach, tier II LLNAs have to be conducted only in cases for which skin irritation potential is in the range of LN activation potential and no structure-activity relationship data indicating a contact allergenic hazard are available.

Administration, Topical↗

Influence of irritants on lymph node cell proliferation and the detection of contact sensitivity to metal salts in the murine local lymph node assay.

Dimethyl sulfoxide (DMSO) and sodium lauryl sulfate (SLS) are known to cause irritation of the skin, and to enhance the penetration of chemicals into the epidermis. In the present study, the lymph node cell (LNC) proliferative response following exposure to irritants, such as SLS and DMSO, was examined in the murine local lymph node assay (LLNA). Exposure to DMSO or SLS aqueous solution induced a small increase in lymph node cell proliferation compared with aqueous solution alone. Exposure to SLS in DMSO caused a significant increase in LNC proliferation. Further, the effect of addition of the irritants in a vehicle on the detection of contact sensitivity to metal allergens was examined. Application of potassium dichromate and nickel sulfate in DMSO or SLS aqueous solution caused increases in LNC proliferation. Exposure to metal allergen with SLS in DMSO also induced a significant LNC proliferative response, but did not induce a significant increase in stimulation index (increase in 3H-thymidine incorporation relative to vehicle-treated control group). This was because of increased 3H-thymidine incorporation following exposure to SLS-DMSO in the control group. These results suggest that irritants enhance the LNC proliferative responses to metal allergens. The use of SLS in aqueous solution is effective for the detection of sensitivity to water-soluble allergens, such as metal allergens, in the LLNA, as well as the use of DMSO as an application vehicle.

Animals↗

Pathologic analysis of sentinel lymph nodes.

The sentinel lymph node (SLN) procedure enables selective targeting of the first draining lymph node, where the initial metastases will form. A negative SLN predicts the absence of tumor metastases in the other regional lymph nodes with a high degree of accuracy. This means that in case of a negative SLN, regional lymph node dissection is no longer necessary. Besides saving patients the significant morbidity associated with lymph node dissection, it will also save costs. Crucial for the success of the SLN procedure is the screening of the SLN for metastases by the pathologist. To this end, several techniques are available such as standard histo- and cytopathological techniques, immunohistochemistry, flow cytometry, and molecular biological techniques. In this paper, the value of these methods for detecting SLN metastases is discussed. Some of these techniques have also appeared to be quite useful for intraoperative evaluation of SLNs. The standard protocol for detection of SLN metastases consists of extensive histopathological investigation including stepped sections stained with hematoxylin and eosin (HE) and immunohistochemistry. Intraoperative frozen section analysis and imprint cytology of SLNs have been shown to be reasonably reliable for detecting breast cancer metastases in SLNs. Further studies are necessary to establish the role of multiparameter flow cytometry and sophisticated molecular biological techniques such as reverse transcription polymerase chain reaction (RT-PCR) in detecting SLN metastases.

Biopsy, Needle↗

Lymph-borne chemokines and other low molecular weight molecules reach high endothelial venules via specialized conduits while a functional barrier limits access to the lymphocyte microenvironments in lymph node cortex.

Lymph-borne, soluble factors (e.g., chemokines and others) influence lymphocyte recirculation and endothelial phenotype at high endothelial venules (HEVs) in lymph node cortex. Yet the route lymph-borne soluble molecules travel from the subcapsular sinus to the HEVs is unclear. Therefore, we injected subcutaneously into mice and rats a wide variety of fluorophore-labeled, soluble molecules and examined their distribution in the draining lymph nodes. Rather than percolating throughout the draining lymph node, all molecules, including microbial lipopolysaccharide, were very visible in the subcapsular and medullary sinuses but were largely excluded from the cortical lymphocyte microenvironments. Exclusion prevailed even during the acute lymph node enlargement accompanying viral infection. However, low molecular mass (MW) molecules, including chemokines, did gain entry into the cortex, but in a very defined manner. Low MW, fluorophore-labeled molecules highlighted the subcapsular sinus, the reticular fibers, and the abluminal and luminal surfaces of the associated HEVs. These low MW molecules were in the fibers of the reticular network, a meshwork of collagen fibers ensheathed by fibroblastic reticular cells that connects the subcapsular sinus floor and the HEVs by intertwining with their basement membranes. Thus, low MW, lymph-borne molecules, including chemokines, traveled rapidly from the subcapsular sinus to the HEVs using the reticular network as a conduit.

Animals↗

Lymph node dissection.

Cervical lymph nodes are involved in 43% of patients with an upper esophageal lesion, 33% of patients with a middle third tumor, and 29% with a tumor of the lower third. Conventional two-field lymph node dissection removing the abdominal and lower mediastinal lymph node groups leads to inaccurate staging and is inadequate for preventing local recurrence. Three-field lymphadenectomy involves bilateral removal of the lower cervical groups of nodes, the superior, middle, and inferior mediastinal lymph nodes and the abdominal groups. The advantages of this extended dissection are improved survival, diminished incidence of local recurrence, and more accurate staging. The benefits in terms of improved survival may accurate to patients both with and without cervical lymph node involvement. Three-field node dissection improves 5-year survival to around 35% without an increase in the mortality rate. The incidence of recurrent laryngeal nerve paralysis is increased with the operation and could be as high as 14% or more.

Carcinoma, Squamous Cell↗

[Methods of sentinel lymph node mapping].

Sentinel lymph node mapping is used as a diagnostic method in order to increase staging accuracy without the well-known morbidities of radical lymphadenectomy. The sentinel node is defined as the first node to receive lymphatic drainage from a primary tumor. The sentinel node concept postulates that if the first draining node is negative for metastasis, the remaining lymph nodes in the nodal basin can be spared. Thus one can predict the status of the nodal basin with high accuracy. It also shows that lymphatic spread seems to follow certain rules that do not concern the whole lymphatic basin. Skip metastases were proven to be rare in breast and gastric cancer. The main issue in sentinel lymph node mapping remains the improvement of staging accuracy for detection of lymph nodes most likely to develop metastasis as well as reducing morbidity by planning lymphadenectomies individually for each tumor and each patient.

Colloids↗

Laparoscopic lymph node dissection around the inferior mesenteric artery for cancer in the lower sigmoid colon and rectum: is D3 lymph node dissection with preservation of the left colic artery feasible?

BACKGROUND: When we perform laparoscopic lymph node dissection around the inferior mesenteric artery (IMA), we preserve the left colic artery (LCA) to maintain the blood supply to the proximal sigmoid colon. In this study, we present our laparoscopic D2 and D3 lymph node (LN) dissection technique and evaluate its applicability and safety. METHODS: We performed LN dissection on 23 rectal and lower sigmoid colon cancer cases from April 2002 to December 2004. For D3 LN dissection, the incision to the mesosigmoid extends to just before the root of the IMA, which is exposed with an ultrasonic cutting and coagulating surgical device to avoid bleeding. Then, the arterial wall is exposed with a dissecting electrocautery spatula down to the LCA, at least 2 cm of which is exposed. Adipose tissue surrounding the IMA and inferior mesenteric vein is dissected. For D2 LN dissection, we partially expose the IMA to confirm the location of the LCA. RESULTS: The mean times taken for D2 and D3 LN dissections were 36.2 and 68.2 min, respectively. Both procedures took longer in male patients. There was a trend for the procedure overall to take less time in female patients. However, D2 dissection took significantly longer in male than female patients (p < 0.05). In women, D3 dissection took significantly longer than D2 (p < 0.05), but this trend was not seen in men. Increased experience among surgeons with this procedure was associated with significantly faster LN dissections in men (p < 0.05), but not in women (p = 0.493). Pearson product moment analysis identified a relationship between body mass index (BMI) and the time taken for D2 LN dissection (r = 0.765), but not D3 LN dissection (r = 0.158). There was no treatment-related morbidity with this technique. CONCLUSIONS: This method was safe and feasible for all patients in this series, but takes longer to perform in male patients.

Aged↗

Is an increase in CD4/CD8 T-cell ratio in lymph node fine needle aspiration helpful for diagnosing Hodgkin lymphoma? A study of 85 lymph node FNAs with increased CD4/CD8 ratio.

BACKGROUND: An elevated CD4/CD8 T-cell ratio on flow cytometry (FCM) analysis has been reported in the literature to be associated with Hodgkin lymphoma (HL). The purpose of our study was to determine the diagnostic significance of an elevated CD4/CD8 ratio in lymph node fine needle aspiration (FNA) specimens. DESIGN: Between 1996 and 2002, out of 837 lymph node FNAs submitted for flow cytometry analysis, 85 cases showed an elevated CD4/CD8 ratio, defined as greater than or equal to 4, without definitive evidence of a lymphoproliferative disorder. The cytologic diagnoses of these 85 cases were grouped into four categories: reactive, atypical, Hodgkin lymphoma (HL), and non-Hodgkin lymphoma (NHL). Histologic follow-up was available in 17/85 (20%) of the cases. RESULTS: 5 of the 64 cases in which FCM and cytology did not reveal evidence of a lymphoproliferative disease had tissue follow-up because of persistent lymphadenopathy and high clinical suspicion. 3/5 (60%) confirmed the diagnosis of reactive lymphadenopathy. The two remaining cases (40%) were positive for lymphoma (1HL, 1NHL). 8/15 cases called atypical on cytology had histologic follow-up. 7/8 (87.5%) cases were positive for lymphoma (3HL, 4NHL). 3/4 cases called HL on cytology had tissue follow-up and all 3 (100%) confirmed the diagnosis of HL. One case diagnosed as NHL on cytology was found to be a diffuse large B-cell lymphoma. In summary, out of 17 cases with histologic follow-up 4/17 (24%) were reactive with CD4/CD8 T-cell ratio of 4.1-29, 7/17 (41%) were HLs with CD4/CD8 T-cell ratio of 5.3-11, and 6/17 (35%) were NHLs with CD4/CD8 T-cell ratio of 4.2-14. CONCLUSION: An elevated CD4/CD8 ratio on FCM is a nonspecific finding which may be seen in both reactive and lymphoproliferative disorders. The cytomorphologic features of the smear are more relevant than the sole flow cytometric finding of an elevated CD4/CD8 ratio.

Journal Article↗

Axillary staging using positron emission tomography in breast cancer patients qualifying for sentinel lymph node biopsy.

Axillary lymph node dissection (ALND) is the standard of care for nodal staging of patients with invasive breast cancer. Due to significant somatic and psychological side effects, replacement of ALND with less invasive techniques is desirable. The goal of this study was to evaluate the clinical usefulness of axillary lymph node (ALN) staging by means of positron emission tomography (PET) with 18F-fluorodeoxyglucose (FDG) in breast cancer patients qualifying for sentinel lymph node biopsy (SLNB). FDG-PET was performed within 1 week before surgery in 24 clinically node-negative breast cancer patients with tumors smaller than 3 cm. Sentinel lymph nodes (SLNs) were identified by preoperative lymphoscintigraphy following peritumoral technetium 99m-labeled colloid albumin injection, and by intraoperative gamma detector and blue dye localization. Following SLNB, a standard ALND was performed. Serial sectioning and immunohistochemistry of the SLN as well as standard histologic examination of the non-SLN was performed. FDG-PET detected all primary breast cancers. Staging of ALNs by PET was accurate in 15 of 24 patients (62.5%), whereas PET staging was false negative in 8 of 10 node-positive patients and false-positive in 1 patient. The sensitivity, specificity, positive predictive value, and negative predictive value of FDG-PET for nodal status was 20%, 93%, 67%, and 62%, respectively. The mean diameter of false-negative ALN metastases was 7.5 mm (range 1-15 mm). Lymph node staging using FDG-PET is not accurate enough in clinically node-negative patients with breast cancer qualifying for SLNB and should not be used for this purpose.

Adult↗

The contemporary incidence of lymph node metastases in prostate cancer: implications for laparoscopic lymph node dissection.

The incidence of lymphatic metastases in 229 consecutive patients with clinically localized prostatic cancer was assessed. Only 13 patients had nodal metastases, for an incidence of 5.7%. A monoclonal prostatic specific antigen value of more than 40 ng./ml. correlated with a positive predictive value of 53% for nodal metastases. Routine laparoscopic node dissection is unnecessary considering the low incidence of nodal metastases.

Humans↗

Adenovirus vector-mediated gene transfer to regional lymph nodes.

Regional lymph nodes (RLNs) possess important immune functions and represent a major pathway of metastasis for solid tumors. Given these facts, the ability to transfer exogenous genes to the RLNs with the goal of manipulating the local immunological milieu would be desirable. On the basis of the hypothesis that a significant proportion of adenovirus (Ad) gene transfer vectors traffic through the lymphatics, E1-E3- Ad vectors were injected into the hind footpad of C3H/He mice and the RLNs assessed for vector trafficking and transgene expression. A low dose (10(9) particles) of an Ad vector encoding the firefly luciferase gene (Ad-CMV.Luc) resulted in luciferase expression only in the injection site and RLNs, with no detectable systemic (liver, spleen, lung) expression. At a higher dose (10(11) particles), some expression could be detected systemically in addition to the RLNs, but at levels in liver 14-fold less than in the RLNs. Transgene expression in the RLNs was transient, peaking at 1 day, decreasing markedly by 7 days. At high doses (10(11) particles), interruption of draining lymphatics decreased the amount of systemic dissemination 22-fold, suggesting that a large proportion of the vector trafficks through the lymphatics before reaching the systemic circulation. Administration of a vector encoding the jellyfish green fluorescent protein gene (AdCMV.GFP, 10(11) particles) showed that transgene expression in the RLNs was primarily in the cortical area. After footpad injection of a fluorescent-labeled Ad vector (Cy3-AdCMV.Null), fluorescent virions were visualized in the draining lymph. Regional lymph collected from animals injected in the footpad with AdCMV.Luc (10(11) particles) contained functional vector. Augmentation of local immune function in the RLNs was achieved by footpad administration of an Ad vector encoding murine IL-12, resulting in high mIL-12 and IFN-gamma levels in the regional, but not distant, nodes. These data demonstrate that expression of exogenous genes in RLNs is easily accomplished with Ad vectors, Ad vector dissemination occurs primarily via the lymphatics after footpad administration in mice, and basic immune functions in the RLNs can be manipulated by Ad-mediated gene transfer in vivo.

Adenoviridae↗

[Could lymph node microangiopathy play a part in the immunodeficiency of diabetes? Histological and immunohistochemical study of lymph nodes from 4 patients with long-term insulin-dependent diabetes mellitus subjected to autopsy examination].

The reason of increased susceptibility to frequent and protracted infections in diabetic patients is still unclear, although a multitude of "in vitro" studies have focused on the metabolic and functional modifications of the immune cells. From the literature immune abnormalities have been demonstrated "in vitro models" in genetic (type 1), autoimmune (type 2) and metabolic (type 1 and type 2) insulin-dependent diabetes mellitus (IDDM), and concisely referred in this paper. Diabetes microangiopathy, which is a peculiar alteration of the disease has been extensively described on many sites such as retina, renal glomeruli, skin etc, but as far as we know, not yet in the lymph-nodes. Therefore we report our histological and immunohistochemical observations in lymph-nodes removed from four patients with long-term IDDM, submitted to autoptic examination. On the clinical history all had shown, in the last years, lymphocytopenia and several infective diseases. The peculiar microangiopatic modifications appear joint to lymphodepletion of B and T cell dependent areas with marked reduction of follicular dendritic reticulum cells. The microangiopathy of thin intranodal capillaries besides compromise the diapedesis and traffic of the T and B lymphocytes could prevent the transformation of endothelial cells into dendritic reticulum cells. Indeed from the recent literature the dendritic reticulum cells should appear derived from transformed endothelial cells. This histological and immunohistochemical report could thence provide some additional knowledge about the complex problem of the immunodeficiency in diabetic patients.

Adult↗