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Background Gall bladder cancer (GBC) is an aggressive cancer with specific predilection like female gender and specific geographical areas, however the molecular mechanisms and factors contributing to the clinical or biological behavior are not understood. Aim The aim of this study was to perform a comprehensive analysis of differentially expressed genes in advanced GBC and chronic cholecystitis (CC) cases. Materials and methods Microarray was planned on fresh specimens of advanced GBC and CC cases using single color cRNA based microarray technique (8X60K format; Agilent Technologies, USA). Twelve advanced GBC and four CC patients were included in the study. Results Of the total of 1307 differentially expressed genes, 535 genes were significantly upregulated, while 772 genes were significantly downregulated in advanced GBC vs CC samples. Differentially expressed genes were associated with biological processes (55.03%), cellular components (31.48%), and molecular functions (13.49%) respectively. The important pathways or key processes affected were cell cycle, DNA replication, oxidative stress, gastric cancer pathway. Using in silico analysis tools, three differentially expressed genes i.e. TPX2, Cdc45 and MCM4 were selected (for their significant role in DNA replication and microtubule function) and were further validated in 20 advanced GBC cohort by immunohistochemistry. Significant positive association of Cdc45 and MCM4 proteins was found in advanced GBC cases (p = 0.043), suggesting the probable oncogenic role of Cdc45 and MCM4 proteins in advanced GBC. Conclusion Our data demonstrate the potential regulation of Cdc45-MCM4 axis in advanced GBC tumors. Additionally, our study also revealed a range of differentially expressed genes (e.g. TPX2, AKURA etc.) between GBC and CC, and further validation of these genes might provide a potential diagnostic or therapeutic target in future.

The recently introduced Revised International Staging System (R-ISS) for multiple myeloma (MM) integrates albumin, β2 microglobulin, lactate dehydrogenase (LDH) with high-risk cytogenetic aberrations (CA), i.e., t(4;14) and t(14;16) and del17p using fluorescent in situ hybridization (FISH). We evaluated utility of nucleic acid-based tests of multiplex ligation-based probe amplification (MLPA) and quantitative real-time polymerase chain reaction (qRT-PCR) to define the CA and the R-ISS categories as per this approach were evaluated for their ability to predict outcome in terms of response, progression-free (PFS), and overall survival (OS). In this study (n = 180), 17 (9.4%), 118 (65.6%), and 45 (25%) patients were assigned to R-ISS1, R-ISS2, and R-ISS3 categories with statistically significant differences in median PFS (p = 0.02) and OS (p 0.001).On univariate analysis, serum creatinine, LDH, 17p deletion, chromosome 1q gain, and response after first induction therapy were associated with statistically significant differences (p 0.05) in PFS and in addition, age 65 years and use of triplet therapy with OS. On multivariate analysis, only serum creatinine, LDH, and response after first induction therapy retained significance for predicting PFS and in addition, use of triplet therapy retained significance for the OS. The proposed nucleic acid-based algorithm using qRT-PCR and MLPA for R-ISS is resource-effective in terms of small quantities of sample required; feasibility of batch processing and reduced overall cost for the total number of regions evaluated and retained the prognostic significance of R-ISS, making it suitable for clinical practice for molecular characterization of MM.

Exploration and sustainable utilization of wild crops are essential for the dietary diversification and also for ensuring the nutritional requirements of growing human population. In this background, the present study was undertaken to evaluate the occurrence, distribution and habitat characterization of two underutilized perennial leafy vegetables of Basellaceae, i.e., Basella alba and Basella rubra, commonly called as Indian spinach or Malabar spinach. For this, extensive field surveys and habitat analysis have been conducted in selected districts of North and West India and national and global distribution maps were prepared based on the available literature. The habitat analysis clearly indicates that Basella species can luxuriously grow in diverse soil conditions (i.e., from acid to alkaline conditions and also in degraded and nutrient poor soils). However, the fresh leaves and stems of both Basella species are rich in protein, vitamin A, vitamin C, Ca, Fe, Mg, P, K, Na, Zn, Cu, Mn and Se and also having essential amino acids and flavones. It can be directly used in salads or can be used as a standalone vegetable for making soups, stews, steamed and oil fried items. In India, it is also being used to make snacks (pakoda). Though it was widely cultivated in Southeast Asia and China since ancient times, it is being gradually neglected and replaced by other greens. Owing to its adaptability to grow luxuriously in hot and humid tropical climate, it can be exploited as a promising leafy vegetable for the warming climatic conditions. However, suitable agronomic practices and crop improvement programs are necessary for improving the adaptability and nutritional quality of Basella species.

The use of novel agents for the treatment of multiple myeloma (MM) has enabled attainment of deeper responses and the information that progression-free survival (PFS) and overall survival (OS) corr...

Purpose To report the long-term control and toxicity outcomes of patients with clinically localized prostate cancer, who underwent low-dose-rate prostate brachytherapy with magnetic resonance spectroscopic image (MRSI)–directed dose escalation to intraprostatic regions. Methods and Materials Forty-seven consecutive patients between May 2000 and December 2003 were analyzed retrospectively. Each patient underwent a preprocedural MRSI, and MRS-positive voxels suspicious for malignancy were identified. Intraoperative planning was used to determine the optimal seed distribution to deliver a standard prescription dose to the entire prostate, while escalating the dose to MRS-positive voxels to 150% of prescription. Each patient underwent transperineal implantation of radioactive seeds followed by same-day CT for postimplant dosimetry. Results The median prostate D90 (minimum dose received by 90% of the prostate) was 125.7% (interquartile range [IQR], 110.3–136.5%) of prescription. The median value for the MRS-positive mean dose was 229.9% (IQR, 200.0–251.9%). Median urethra D30 and rectal D30 values were 142.2% (137.5–168.2%) and 56.1% (40.1–63.4%), respectively. Median followup was 86.4 months (IQR, 49.8–117.6). The 10-year actuarial prostate-specific antigen relapse–free survival was 98% (95% confidence interval, 93–100%). Five patients (11%) experienced late Grade 3 urinary toxicity (e.g., urethral stricture), which improved after operative intervention. Four of these patients had dose-escalated voxels less than 1.0 cm from the urethra. Conclusions Low-dose-rate brachytherapy with MRSI-directed dose escalation to suspicious intraprostatic regions exhibits excellent long-term biochemical control. Patients with dose-escalated voxels close to the urethra were at higher risk of late urinary stricture.

In chronic lymphocytic leukemia (CLL), the geographical bias in immunoglobulin heavy-chain variable (IGHV) gene usage lead us to analyze IGHV gene usage and B-cell receptor stereotypy in 195 patients from India. IGHV3, IGHV4, and IGHV1 families were the most frequently used. 20.5% sequences had stereotyped BCR and were clustered in 12 pre-defined and 6 novel subsets. Unmutated IGHV was significantly associated with reduced time to first treatment (p

Background: Tympanoplasty involves reconstitution of the tympano-ossicular system with commonly. fascia of the temporalis muscle, situated in its proximity. The fascia is grafted on the residual tympanic membrane by placing it either over it or below it, after creating a raw surface. The former is the overlay and latter, the underlay technique. In this study on restitution of the ear drum utilizing the overlay and underlay techniques, an exhaustive analysis of the two modalities has been done, taking into consideration various variables individually. The surgical outcome wrt to graft uptake and hearing gain has been compared in "depth" with extensive studies undertaken in India and abroad. The unbiased tabulated comparison of each aspect is unique and would guide future researchers to opt the ideal modality.Methods: Tympanoplasty was undertaken in chronic safe suppurative otitis media with the underlay and overlay techniques in a study group of 40 patients in this prospective study. The patients were randomly divided into equal groups for either procedure. Results: In the cohort of 40 subjects successful graft uptake was observed in 16 (80%) with overlay technique and 17 (85%) with underlay technique. Graft rejection was reported in 4 (20%) and 3 (15%) cases with overlay and underlay techniques respectively. Hearing improvement was 56.25% in 10-20 dB range in Overlay. Whereas, with underlay it was 47.05% in 10-2 dB.Conclusions: Overlay technique is ideal for anterior and central perforations while underlay for subtotal and posterior perforations. In terms of hearing improvement, the fibrosis during graft uptake makes the results of either technique unpredictable.

Complex Chromosomal Rearrangements (CCRs) are increasingly being reported as genetic risk factors of clinical significance in cancer owing to their identification using high resolution whole genome profiling technologies. This study employed high resolution CGH + SNP microarrays for whole genome copy number variations (CNV) profiling and identified CCRs in 11/107(10%) newly diagnosed Multiple Myeloma (MM) patients. Six patients exhibited Chromothripsis (CTH) among seven chromosomes that were confirmed with automated CTLPscanner web tool and; five cases displayed chromoplexy (CPL) which involved multiple chromosomes. Presence of chromothripsis in chromosome 17 in three out of six patients indicate a link between TP53 aberrations and incidence of CTH. Multivariable Cox regression model demonstrated a significant association of CTH with poor PFS (HR = 3.09, p = 0.010) and OS (HR = 3.31, p = 0.024) which suggests that CTH is an additional independent prognostic marker in multiple myeloma. Addition of CTH in risk stratification models in clinical setting in multiple myeloma may help in upfront identification of high risk patients for suitable customized therapy.

T regulatory cells (Tregs) (CD4 + CD25 + cells) have immunosuppressive function and constitute 5–10% of all peripheral CD4 + lymphocytes. T-helper 17 (TH17) cells are new effector T-Helper cells th...

Serum vascular endothelial growth factor (VEGF) level is higher in children with non-Hodgkin lymphoma (NHL) than healthy controls, but its prognostic significance is unknown [1]. VEGF expression ha...

PURPOSE: Rates of rectal toxicity after low-dose-rate brachytherapy for prostate cancer are dependent on rectal dose which is associated with rectal distance from prostate and implanted seeds. Placement of a hydrogel spacer between the prostate and rectum has proven to reduce the volume of the rectum exposed to higher radiation dose levels in the setting of external beam radiotherapy (EBRT). We present our findings with placing a rectal hydrogel spacer in patients following low dose-rate LDR brachytherapy, and we further assess the impact of this placement on dosimetry and acute rectal toxicity. MATERIALS AND METHODS: Between January 2016 and April 2017, 74 patients had placement of a hydrogel spacer, immediately following a Pd-103 seed-implant procedure. Brachytherapy was delivered as follows: as a monotherapy to 26 (35%) patients; as part of planned combination therapy with EBRT to 40 (54%) patients; or as a salvage monotherapy to eight (11%) patients. Post-operative MRI was used to assess separation achieved with rectal spacer. Acute toxicity was assessed retrospectively using RTOG/EORTC radiation toxicity grading system. Rectal dosimetry was compared with a consecutive cohort of 136 patients treated with seed implantation at our institution without a spacer, using a 2-tailed paired Students’ T-test (p

Purpose Intraoperative radiation therapy (IORT) allows delivery of tumoricidal doses of radiation to areas of potential residual microscopic disease while minimizing doses to normal tissues. IORT using high-dose-rate (HDR) brachytherapy allows dose modulation and delivery of concomitant boosts to high-risk areas. This study describes a novel technique of HDR-IORT with dose painting (DP) (HDR-IORT-DP) and evaluates the clinical outcomes. Methods and Materials Sixteen patients with recurrent cancers received HDR-IORT-DP at the time of radical resection. Of these patients, 13 had colorectal cancer, 2 had head and neck cancer, and 1 had a gynecologic malignancy. All received external beam radiation previously. Negative margin (R0) was obtained in 12 patients (75%) and microscopically positive margins (R1) in 4 patients (25%). Results The median total target and boost area were 45 and 8.5 cm2, and HDR-IORT and boost dose were 1500 and 1750 cGy, respectively. Median followup was 14.9 months. The 2-year local control and overall survival were 80% and 20%, respectively. Eleven patients (69%) developed distant metastasis and were deceased at the time of the last followup. A total of 13 patients (19%) developed Grade 3 toxicity related to HDR-IORT; no grade 4+ toxicities were observed. Conclusions HDR-IORT-DP technique is feasible, safe, and allows for dose escalation in locally advanced or recurrent previously irradiated tumors. To our knowledge, this is the first clinical report on HDR-IORT-DP. Further studies are warranted to evaluate efficacy in a larger patient cohort. Local control was encouraging in our patients.

Despite improved treatment options, recurrence rates remain high in ovarian cancer. Tumor microenvironment, particularly T cells, plays an important role in its natural history. The present study was conducted to study the kinetics of T cell subsets in homogenous population of advanced-stage ovarian cancer patients of serous histology with reference to treatment and outcome. We assessed the frequencies of CD8+ T cells, CD4+ T cells, regulatory T cells (Tregs, CD4+ CD25intermediate to high FOXP3+ CD127low) and natural killer cells in peripheral blood at baseline (n = 41) and post-treatment (n = 27) in the patients and compared it with blood of healthy controls (n = 15). The ascitic fluid of 12 of these patients was compared with ascitic fluid of patients with cirrhotic liver disease (n = 5). Higher numbers of Tregs, CD8+ T cells and higher Treg/CD4 ratio and reduced numbers of CD4+ T cells and reduced CD4/CD8 ratio were observed in patients as compared to controls. An increase in total number of T cells was observed with treatment, while no change in frequency of any lymphocyte subset was evident. These results support previous studies exhibiting an increase in Tregs in ovarian cancer patients. Unchanged levels of T cell subsets subsequent to treatment suggest that they may be resistant to reset to healthy control homeostatic levels with standard chemotherapeutic treatments. Lower CD4 levels, higher CD8 levels, altered CD4/CD8 ratio and T cell subsets call for evaluation of role of immunomodulating agents or Treg-targeted immunotherapy in the treatment of advanced-stage serous carcinoma ovary.

Objectives: In bone marrow, hematopoietic stem cells (HSCs) reside in the most hypoxic endosteum niche, whereas the proliferating progenitors are located near the relatively oxygen-rich vascular region. High oxygen tension is potentially detrimental to HSCs. The objective of this investigation was to compare cellular, functional, and molecular responses of human umbilical cord blood (UCB)-derived hematopoietic stem and progenitor cells in culture under hypoxic and normoxic conditions. Methods: CD133-enriched UCB cells were cultured in growth factor containing serum-free and serum-supplemented medium under 5% O2 (hypoxia) or 21% O2 (normoxia) for 10 d. The phenotypes of expanded cells were analyzed by flow cytometry and the engraftability by SCID-repopulation assay. The expression of hypoxia-inducible factor (HIF)-1α and some of its target genes was analyzed by real-time RT-PCR. Results: In hypoxic culture, CD34+ CD38− cells were expanded about 27-fold, which was significantly (P

Comparison of characteristic features, radiology, management and recurrence pattern of fungal sinusitis between children and adults.A prospective study conducted in the department of Otorhinolaryngology, Head and Neck Surgery, Post Graduate Institute of Medical Education and Research, Chandigarh in which all the cases presenting with the features of allergic fungal sinusitis (AFS) between January 2000 and January 2005 were enrolled. These cases were divided into two groups, group 1 comprised of cases with age less than 15 years and group 2 with age more than 15 years. Detailed history, physical examination and nasal endoscopic examination and computed tomography (CT) scan of the paranasal sinuses was done in all the cases. The cases with prior history of sinonasal surgery were excluded from the study. All patients refractory to medical management were subjected to endoscopic sinus surgery. All the cases were followed up for a period ranging from 6 to 39 months to see for the recurrence. The data of both the groups was analysed statistically using chi square test.The study population comprised of 200 cases, with 68 cases in group 1 and 132 cases in group 2. The most common symptom in both the groups was presence of nasal obstruction. The children had higher incidence of having unilateral disease (46 out of 68) compared with adults, where it was 38 out of 132. The bony erosion was seen more often in group 1. Surgery was done endoscopically in all the cases. The intra orbital or intra cranial extension was seen in 58 cases of group 1 and 47 cases of group 2 (p0.001). Recurrence was seen in 18 (15 with intraorbital and 3 with intracranial extension) cases in group 1 and 13 cases (11 with intraorbital and 2 with intracranial extension) in group 2 (p0.005).In our study, we found a higher incidence of facial deformities, proptosis, intraorbital/intracranial extension and a higher rate of recurrence in group 1, therefore, suggesting a more aggressive nature of AFS in children than adults mandating an early diagnosis, proper management and regular follow up in these cases.

In modern digital communication systems, arbitrary sample rate conversion is the most computation intensive task. In addition, a reconfigurable sample rate converter is often required to meet the sampling rate requirements of different radio standards. This paper proposes a pipelined architecture for FPGA implementation of arbitrary rate converter employing cut-set retiming and Sum-Of-Power-Of-Two (SOPOT) techniques to achieve high throughput while reducing the hardware. The proposed architecture for 16 bit precision is designed and implemented using Xilinx ISE 14.2 and XC3S500E-4FG320 FPGA device. The implementation results show that the proposed architecture improves throughput by 4.5 times.

8044Background: Cystatin-C is recently gaining significance as a marker for disease activity in multiple myeloma. We aimed to study whether serum Cystatin-C is prognostic of disease severity and pr...

Inappropriate sinus tachycardia (IST) is a clinical syndrome characterized by presence of non-paroxysmal tachyarrhythmia manifesting with increased resting heart rate and exaggerated or persistent response to exercise or position. When IST is intolerable and medically refractory, invasive therapies such as sinus node modification or atrioventricular node ablation with placement of permanent pacemaker are tried to control symptoms. We present a 34 year old patient with symptomatic IST unresponsive to medical therapy who underwent anesthetic block of the right and left stellate ganglia. At four month follow up the patient demonstrated sustained improvement in heart rate and reported freedom from previous symptoms.

In bone marrow, hematopoietic stem cells (HSCs) reside in the most hypoxic endosteum niche, whereas the proliferating progenitors are located near the relatively oxygen-rich vascular region. High oxygen tension is potentially detrimental to HSCs. The objective of this investigation was to compare cellular, functional, and molecular responses of human umbilical cord blood (UCB)-derived hematopoietic stem and progenitor cells in culture under hypoxic and normoxic conditions.CD133-enriched UCB cells were cultured in growth factor containing serum-free and serum-supplemented medium under 5% O(2) (hypoxia) or 21% O(2) (normoxia) for 10 d. The phenotypes of expanded cells were analyzed by flow cytometry and the engraftability by SCID-repopulation assay. The expression of hypoxia-inducible factor (HIF)-1α and some of its target genes was analyzed by real-time RT-PCR.In hypoxic culture, CD34(+) CD38(-) cells were expanded about 27-fold, which was significantly (P0.01) higher than that obtained in normoxic culture. Serum-free culture did not support the growth of cells in the presence of 21% O(2) . Myeloid colony-forming potential of cells was significantly (P0.05) increased in 5% O(2) compared with 21% O(2) culture. SCID-repopulation efficiency seems to be better preserved in the cells cultured under hypoxic conditions. Hypoxia significantly (P0.05) induced the expression of HIF-1α, vascular endothelial growth factor (VEGF), and ABCG2 genes and also upregulated CXCR4 receptor expression.Low oxygen tension enhanced the proliferation of UCB-derived HSC/progenitor cells and maintenance of SCID-repopulating cells than normoxia. These expanded cells are expected to be beneficial in the patients who lack human leukocyte antigen (HLA)-matched donors.

The application of cord blood (CB)-derived hematopoietic cells in allogeneic transplantation has been restricted because of the unavailability of adequate cell numbers from one unit of CB and their delayed engraftment in recipients. The main purpose of this study was to develop an economic process for isolating nucleated cells from CB and expanding enriched CD133(+) cells.Four protocols for processing CB, using different combinations of density-gradient centrifugation, hydroxyethyl starch (HES) and NH4Cl treatment, were compared regarding the yields of CD45(+), CD34(+)/CD133(+) and colony-forming cells. CD133-enriched cells were expanded for 10 days in the presence of hematopoietic growth factor-supplemented medium. The performance of the culture was evaluated by analyzing colony-forming potential, fold-expansion of primitive hematopoietic stem cells (HSC) and lineage commitment by flow cytometry.A two-step treatment of CB with HES followed by NH4Cl resulted in the highest recovery of CD45(+) (84.3%) and CD34(+) (69.1%) cells compared with that present in umbilical CB. A suspension culture of CD133-enriched cells resulted in an average 150-fold increase in nucleated cells, of which CD133(+), CD34(+) and CD34(+) CD38 cell expansions were 17.2+/-1.3, 40+/-4.6, and 6.9+/-1.1 fold, respectively. The total myeloid colony-forming cell count was almost 3800-fold higher than that present in the processed CB.The highest yields of nucleated and progenitor stem cells were obtained with a two-step processing of CB. The CD133(+) cells obtained by this method are expected to yield enough hematopoietic progenitors for potential allogeneic transplantation.

Cardiac complications are a common cause of death in individuals with the inherited multisystemic disease myotonic dystrophy type 1 (DM1). A characteristic molecular feature of DM1 is misregulated alternative splicing due to disrupted functioning of the splicing regulators muscleblind-like 1 (MBNL1) and CUG-binding protein 1 (CUGBP1). CUGBP1 is upregulated in DM1 due to PKC pathway activation and subsequent CUGBP1 protein hyperphosphorylation and stabilization. Here, we blocked PKC activity in a heart-specific DM1 mouse model to determine its pathogenic role in DM1. Animals given PKC inhibitors exhibited substantially increased survival that correlated with reduced phosphorylation and decreased steady-state levels of CUGBP1. Functional studies demonstrated that PKC inhibition ameliorated the cardiac conduction defects and contraction abnormalities found in this mouse model. The inhibitor also reduced misregulation of splicing events regulated by CUGBP1 but not those regulated by MBNL1, suggesting distinct roles for these proteins in DM1 cardiac pathogenesis. The PKC inhibitor did not reduce mortality in transgenic mice with heart-specific CUGBP1 upregulation, indicating that PKC inhibition did not have a general protective effect on PKC-independent CUGBP1 increase. Our results suggest that pharmacological blockade of PKC activity mitigates the DM1 cardiac phenotype and provide strong evidence for a role for the PKC pathway in DM1 pathogenesis.

The syndrome of heart failure is characterized by increased levels of circulating inflammatory mediators, which have been implicated in the pathogenesis of heart failure. Recently, a number of studies have suggested that statins may exert salutary effects in patients who have heart failure by virtue of their pleiotropic (non–lipid lowering) actions. This article focuses on the non–lipid lowering effects of statins, with an emphasis on the anti-inflammatory properties of these agents.

e22073 Background: The contribution of epigenetic silencing of genes by aberrant methylation has been reported to be important in chronic lymphocytic leukemia (CLL). In the present study, gene meth...

Phosphine has been known to science since the birth of modern chemistry. WHO reports that the technical product usually has a foul odour, like "fishy" or "garlicky" because of the presence of substituted phosphines and diphosphine (P2H4). Many medico-legal autopsy cases have been reported positive for aluminium phosphide even though there was neither any suspicion of consuming aluminium phosphide nor any clinical findings, postmortem findings or circumstantial evidences. The present study was carried out to validate the qualitative test, presently applied in many laboratories for testing phosphine. It was observed that 65% of human tissues in saturated solution of common salt show positivity for phosphine gas on the first day of autopsy.

e15099 Background: Gall bladder cancer (GBC) have predilection for female sex, specific geographical areas, prior history of gall stone disease and carries worse prognosis. The reason for this is n...

Induction of invasion gene transcription and expression of the invasive phenotype of Salmonella strains are regulated by environmental conditions. Experimental evidence indicates that oxygen, pH, and osmotic conditions need to closely resemble those of the host intestinal lumen for invasion gene activation. The hilA gene, encoded on Salmonella pathogenicity island 1 (SPI-1), is a transcriptional activator which is required for invasion and whose expression is modulated by oxygen, pH, and osmolarity. Additionally, hilA is regulated by genetic elements encoded on SPI-1 (hilC/sirC/sprA and hilD), as well as by elements which reside outside of SPI-1 (phoP/phoQ and sirA), although how environmental signals modulate hilA is unknown. In an effort to further characterize the Salmonella invasion gene regulon, we have created and preliminarily characterized 18 Tn5 insertions which result in upregulation of a hilA::lacZY fusion. We have classified the mutations based on location and phenotype into three classes. Six class 1 and six class 2 mutants have insertions in SPI-1 near the invasion gene orgA or the invasion gene regulator hilD, respectively. Six class 3 mutants reside outside of SPI-1 in four different loci. The class 2 and 3 mutations induce overexpression of an episomal hilA::lacZY fusion and significantly increase S. typhimurium invasion of HEp-2 cells in a standard invasion assay. These data implicate new regions of SPI-1 as being involved in the regulation of invasion by S. typhimurium and identify new invasion gene regulators located outside of SPI-1.

Salmonella typhimurium initiates infection of a host by invading M cells of Peyer's patches within the small intestine. The ability of the bacteria to invade mammalian cells has been shown to be regulated by environmental conditions, including oxygen concentrations, osmolarity, and growth phase. We have previously created oxygen-regulated Tn5lacZY S. typhimurium mutants that are defective in invasion. We have now identified the invasion genes disrupted by eight of the transposon insertions. These genes encode transcriptional regulators (hilA and invF), type III secretory components (orgA, invG and spaR) and secreted proteins (invC and invD). Examination of the protein-secretion profiles of the non-invasive mutants indicated that each of the mutants was defective in secretion of between one and six proteins. We have also demonstrated that the loss of tissue culture cell invasiveness corresponds to an inability to invade and destroy M cells of Peyer's patches in a murine ligated loop model. Virulence studies, performed in mice, demonstrated that these defects significantly reduced the ability of the mutants to cause murine typhoid fever by an oral route of infection. Virulence by an intraperitoneal route of infection was unaffected. The data indicate that in vitro invasiveness, invasion-protein secretion, and M-cell invasion are critical indicators of S. typhimurium virulence.