Tuberculosis in Dr. Granville’s mummy

Source: Donoghue HD, Lee OY, Minnikin DE, Besra GS, Taylor JH, Spigelman M.

Tuberculosis in Dr Granville's mummy: a molecular re-examination of the earliest

known Egyptian mummy to be scientifically examined and given a medical diagnosis.

Proc Biol Sci. 2010 Jan 7;277(1678):51-6.

 

As soon as I read the title of this interesting article, I knew that I need to write a blog on it. Luckily the full text of the paper was also available online. This work is a collaborative effort of well known researchers from Centre for Infectious Diseases, University College, London, School of Bioscience, University of Birmingham, Department of Ancient Egypt and Sudan, The British Museum, London and Kuvin Centre for Study of Infectious and Tropical Diseases, The Hebrew University, Israel.

Dr. Augustus Bozzi Granville was an eminent physician and obstetrician who examined an Egyptian mummy by scientific autopsy in 1825. The mummy was of a 50 year old woman named, lady Irtyersenu. She was from necropolis of Thebes, of the 26^th Dynasty dated to 600BC. After detailed observation of the wrappings and the exterior features of the mummy, Granville decided to perform a detailed autopsy. This mummy is commonly called as Granville's mummy. Granville observed a mass around the right ovary, which was described by him as ovarian dropsy or cancer and he proposed that this was the cause of her death. Recent studies have however, shown that this was actually a benign cystadenoma of the ovary, which is described as non-fatal. Other studies carried out recently mentioned the presence of a pulmonary exudate, which can be a potentially fatal pathological condition. Many studies were carried out to determine the exact cause of death of the mummy. During the early 1990s, several attempts were made to detect microbial or human DNA from the Granville mummy by PCR. But these studies were not successful.

Based on paleopathological changes, tuberculosis has long been recognized in Egyptian mummies. Furthermore, M. tuberculosis complex DNA has been detected and characterized from the predynastic era, the Old, Middle and New kingdoms. Thus, authors of this paper sought to determine whether lady Istyersenu had tuberculosis, which might have been the cause of her death. For this they used two strategies. These were detection of M. tuberculosis by PCR and direct detection of specific cell wall mycolic acid biomarkers of the M. tuberculosis complex, using HPLC.

For performing these experiments they used samples taken during 1990 (lung tissue, bone from the right and left femurs and hand) and some additional samples were taken. Special precautions were taken during DNA extraction and PCR procedures.

DNA from the M. tuberculosis complex was detected in lung samples, gall bladder and membranous tissue by PCR, but the results were inconsistent. The authors could not perform additional characterization of M. tb DNA, as this DNA was extremely fragile. However, they performed DNA sequencing from nested PCR products. These sequences were homologous with those of M. tb. The HPLC analysis showed a good correlation between the profiles from samples collected from different sites and presence of specific biomarkers for Tb was clear. The authors thus proposed that assuming that the pulmonary exudate was the prime source of the infection, it appears that the lady Irtyersenu had pulmonary tuberculosis that had disseminated to other sites in the body.

NOD Like Receptors (NLRs) in Human B Cells

We all know that innate immune response provides first line of defense against invading micro-organisms. Cells of the innate immune system express certain proteins that are called pattern recognition receptors (PRRs). These receptors identify microbe associated molecular patterns (MAMP) or pathogen associated molecular patterns (PAMP), which are associated with microbial pathogenesis. These PRRs can be divided into two types based on their presence on/in the cells. These two types include membrane bound PRRs (most famous example is Toll like rector (TLR), and cytoplasmic PRRs.

The NOD like receptors (NLRs) are the cytoplasmic PRRs. The NLR family comprise of 20 intracellular proteins that recognize primarily bacterial structures. They are divided into three subfamilies, NODs, NALPs and NAIPs. The best characterized members of the NOD sub-family are NOD1 and NOD2. NOD1 recognizes a molecule called meso-DAP, specific for Gram negative bacteria and NOD2 recognizes MDP, a component of all types of bacterial peptidoglycans. In the NALP subfamily, NALP1beta and NALP3 are most well-known members. Members of NALP family respond to danger signals released from injured or dying cells, leading to formation of inflammasomes. The members of NAIP family detect intracellular flagellin and also lead to the formation of inflammasome. Expression of NLRs has been detected in many cells including DCs, macrophages, monocytes, epithelial and endothelial cells and osteoblasts. However, at present it is not known whether B cells also express NLRs or not. In the present study, the authors thus investigated the expression of various NLRs and their function in human peripheral and tonsillar B cell subsets. The authors also explored the cooperative roles of NLRs and selected TLRs in B cell activation.

This paper is from the laboratory of Dr. Lars-Olaf Cardell, Division of ENT Diseases, CLINTEC, Karolinska Institute, Sweden and is published in Journal of leukocyte Biology, September 2010 issue.

Petterson T, Jendholm J, Månsson A, Bjartell A, Riesbeck K, Cardell LO. Effects of NOD-like receptors in human B lymphocytes and crosstalk between NOD1/NOD2 and Toll-like receptors. J Leukoc Biol. 2010 Sep 15.

 

A summary of major results obtained by the investigators is as follows:

• Expression of NLRs in human B lymphocytes: Freshly isolated peripheral and tonsillar and B lymphocytes (from patients) were used and mRNA expression of NOD1, NOD2, NALP1, NALP3, NAIP, and IPAF was analyzed using real-time PCR. The results of the PCR assay showed that peripheral B cells expressed all the NLRs tested in this experiment, except IPAF. In contrast, tonsillar cells only expressed NOD1, NALP1, and NAIP. Furthermore, flow cytometric and immunohistochemistry analyses were done to confirm that results of real-time PCR. For these analyses, rabbit polyclonal Ab against NOD1 and NAIP and mouse mAb against NOD2, NALP1 and NALP3 were used. It was observed that peripheral B cells expressed all the tested receptors, while tonsillar B cells showed expression of NOD1, NALP1 and NAIP.
  
• B cell receptor triggering is required for NLR-induced B cell activation: To determine whether NLRs have the capability to induce B cell activation upon stimulation with their ligands, purified B cells were isolated and cultured with or without iE-DAP (NOD1 ligand) and MDP (NOD2 ligand) in the presence or absence of BCR i.e., alpha IgM or IgD-binding protein MID. It was observed that when B cells were cultured with NOD1/2 ligands only, no proliferation was observed. However, when cells were stimulated with NLR-ligands in combination with BCR cross-linking via IgM or IgD, a profound increase in B cell proliferation was observed. To examine the role of physical T cell help in NOD induced B cell proliferation, the authors added rCD40L to highly purified B cells. They observed that CD40L in combination with the NLR ligands did not lead any additional proliferation of B cells. To find out more about NLR-induce B cell activation, the investigators stimulated B cells with NOD1/2 ligands together with BCR cross-linking (IgM/IgD) and analyzed expression of cell surface glycoproteins and viability. The number of CD27+, CD69+, CD71+, CD80+, CD86+, and CD95+ B cells was found to be increased. In peripheral B cells, the expression of all these markers were found to be elevated in comparison to unstimulated cells or to those stimulated with BCR alone. In tonsillar B cells, only stimulation with NOD1 ligand increased the percentage of CD71+, CD80+, CD86+, and CD95+ cells. It was also observed that viability of B cells cultured with NOD1 ligands along with BCR was slightly enhanced than controls. However, culture with NOD2 ligands along with BCR did not cause any increase in viability. As tonsillar B cells showed lower B cell responses, the next question was why? To answer this question, investigators determined the level of NLR proteins in naïve (CD19+IgD+CD38-), germinal center (CD19+IgD-CD38+) and memory (CD19+IgD-CD38-) B cells. However, they could not find any difference between blood derived and tonsillar cells.
  
• NLR stimulation augments TLR-induced B cell proliferation: To study the interplay between NLRs and TLRs on B cells, the investigators incubated B cells with suboptimal concentration of TLR agonists in combination with NOD1/2 ligands and BCR cross-linking. It was observed that proliferation of peripheral B cells was enhanced, when they were incubated with TLR agonists. In contrast, only co-stimulation with NOD1 ligand increased the proliferation in tonsillar B cells.
  

Conclusions:

1. This is the first study to demonstrate the presence of a range of NLRs in purified human B cells.
   
2. The study shows that peripheral B cells express NOD1, NOD2, NALP1, NALP3 and NAIP, while tonsillar B cells express NOD1, NALP1 and NAIP.
   
3. Activation of B cells by NLR ligands required BCR cross linking and did not require T cell help.
   
4. NLRs enhanced TLR-induced activation of B cells.

CTLA-4 controls regulatory T cell peripheral homeostasis and is required for suppression of pancreatic islet autoimmunity

CTLA-4 is a member of the immunoglobulin super-family. It is expressed on the surface of helper T cells and transmits an inhibitory signal to T cells. Earlier studies have linked polymorphisms in CTLA-4 gene with several autoimmune diseases including type1 diabetes, thyroiditis and SLE. Severe disease has been observed in CTLA-4 deficient mouse. Several reasons have been proposed for this, although the exact is not known. These reasons include the ability of CTLA-4 to raise the threshold of T cell activation, to modulate T cell motility, alter T cell thymic selection or control the function of regulatory T cells (Tregs). Much work has been done to identify the role of CTLA-4 in Tregs function.

This paper is from the labs of Dr. Lucy. S. Walker, University of Birmingham Medical School, Birmingham, UK. The authors have tried to analyze the role of CTLA-4 in Tregs and the results are as follows:

• Treg are over-expressed in CTLA4-/- mice: To quantify Tregs in CTLA4 knock out mice (CTLA4-/-), authors used intracellular Foxp3 staining. Foxp3 (Forkhead box P3) is a specific marker for Tregs. The authors observed that Foxp3+ Tregs were over-expressed in CTLA4-/- mice in both peripheral LN and spleen in comparison to controls.
  
• 
  CTLA4-/-Treg show unaltered thymic selection but increased peripheral proliferation: The increased expression of Tregs in CTLA4-/- mice can possibly indicate thymic selection or enhanced proliferation in the periphery. To answer these questions, the authors stained thymus cells for CD4, CD8 and Foxp3. They found that the proportion of CD4+Foxp3+ cells was not much different in two groups of mice. To determine whether there was an increased proliferation of Tregs in CTLA4-/- mice, spleen sections from CTLA4-/- animals and controls were stained for CD4, Foxp3, and Ki67 (nuclear protein expressed by proliferating cells). The results showed increased proliferation of Foxp3+ cells in CTLA4-/- mice compared to controls.
  
• CTLA4 regulates proliferation of OVA-specific Treg: Interpretation of data obtained from CTLA4-/- animals is complicated by the lethal lymphoproliferative syndrome that they develop. To overcome this, CTLA4-/- mice can be bred onto a TCR transgenic background (DO11.10), directly their specificity to a non self antigen (OVA). These animals can further be maintained in a rag deficient background. Such DO11/CTLA4-/-/rag-/- mice do not develop lymphoproliferative syndrome and their T cells bear a naïve phenotype. But the DO11/rag-/- animals do not develop Treg. The authors therefore crossed these mice with those that expressed OVA under the control of RIP. This strategy allowed the authors to study Treg in mice that lack CTLA4 but have an intact CD28. In DO11+ mice that did not express the RIP-mOVA ag, DO11 T cells did not develop into Tregs. In antigen positive mice, a fraction of DO11+ T cells developed into Tregs. Thus, CTLA4 signaling is not an obligate step in Treg differentiation. The authors next examined whether CTLA4 expression limited Treg proliferation in the periphery. They observed that Treg in peripheral lymphoid organs of DO11xRIP-mOVA/rag-/- mice remained largely undivided even in the absence of CTLA4. In contrast, Tregs isolated from Pancreas exhibited greater proliferation in mice that lacked CTLA4. All these findings support the idea that CTLA4 signaling controls Treg proliferation in response to encounter with self antigens.
  
• Ab-mediated CTLA4 blockade augments Treg proliferation in BALB/c mice: The authors next tested whether Ab-mediated CTLA4 blockade in normal animals enhanced Treg proliferation, as shown in CTLA4 knockout mice. Mice, which were treated with anti-CTLA4 mAb showed increased ki67 within CD4+Foxp3+ cells after 8 days. Three days following blocking, Foxp3+ cells (Tregs) showed significantly increased proliferation than controls (treated with control mAb). The proliferation of Foxp3- T cells was not much different from that seen in control mAB treated mice. By day 8, Foxp3- cells showed an increased proliferation in anti-CTLA4 Ab treated mice, but this increase became significant only at day 14.
  
• Ag-specific Treg deficient in CTLA4 fail to suppress autoimmune diabetes: The results of investigators raised the paradox that a fatal lymphoproliferative syndrome develops in the CTLA4 knockout mice despite an increase in Treg. To resolve this paradox the authors hypothesized two reasons. Firstly, CTLA4 deficiency might render Treg pathogenic and secondly, CTLA4 might be required for Treg suppression. To test the first hypothesis, authors utilized the fact that injection of CTLA4-/- lymphocytes into rag deficient recipient mice can transfer lymphoproliferative syndrome. Thus, they assessed the ability of purified CD4+CD25- or CD4+CD25high cells from CTLA4-/- to transfer this disease. It was observed that introduction of CD25- cells from CTLA4-/- mice induced the lymphoproliferative syndrome while recipients of CD4+CD25+high cells from CTLA4-/- mice remained healthy. To test the second possibility that CTLA4 is necessary for Treg suppression in vivo, the authors took advantage of the fact that Ag-specific Treg lacking CTLA4 can be purified from TCR transgenic mouse. CD4+CD25+ cells were isolated from DO11xRIP-mOVA/rag-/- mice that were either CTLA4 sufficient or deficient. Treg proportion was equivalent between CTLA4 deficient or sufficient groups. To test the ability of these Tregs to control diabetes in an adoptive transfer model, disease was induced by transfer of OVA-specific CD25- cells into mice expressing OVA in the pancreas and then Tregs from both groups were introduced. It was observed that mice that received CTLA4 sufficient Treg were completely protected from diabetes, while mice that received CTLA4 deficient Treg were not protected from diabetes. To further explore the basis for the failure of CTLA4-/- Treg to control diabetes, the investigators analyzed the expression of IL-10 and surface TGF-beta. No difference in IL-10 expression was observed between wild type and CTLA4-/- Tregs. The expression of TGF-beta was higher in CTLA4-/- Tregs. The investigators further investigated the hypothesis that Treg-expressed CTLA4 can deplete antigen presenting cells of co-stimulatory ligands. The authors found that the presence of Treg resulted in marked down-regulation of CD86 expression on APC and this down-regulation could be blocked by using anti-CTLA4 Ab. Expression level of MHC class II on APC was unaltered by Treg. Taken together, these data suggest that Treg-expressed CTLA4 plays important role in the regulation of an anti-islet immune response and this reflects a capacity for Treg to influence APC in a CTLA4 dependent manner.
  

 

Is human papilloma virus viral load a clinically useful predictive marker: a longitudinal study

Human papilloma viruses (HPV) are a group of more than 100 related viruses and they are one of the primary causes of cervical cancer in women. These are divided into low risk and high risk types based on their ability to cause cancer. There is need to distinguish between those women who test positive for high risk (HR) human papilloma virus (HPV) types and are likely to acquire cervical neoplasia and those women who test positive for HR-HPV but who are not at increase risk. This distinction would require one to study biomarkers. One such biomarker is HPV viral load. It has been observed that in women who are positive for HR HPV types, cytological abnormalities were found to be positively associated with HPV viral load. However, the relationship between HPV viral load and cervical neoplasia is not clear and seems to be complex. In this study, the authors have tried to determine this relationship by measuring viral load of HPV16 and HPV18, HR-HPV types in serial samples taken during the follow up of a group of young women who were recruited soon after they first had sexual intercourse.

Results:

Viral load waxes and wanes during follow up: In the study, 118 women who had an incident of HPV infection (HPV16 or HPV18) as detected by GP5+/GP6+ system and who provided at least three samples for qPCR during follow-up, at least one of which was positive, were studied. Sixty women were tested positive for HPV16 using qPCR. Of these 60, 41 tested positive in all three samples; 10 in two; and 9 in one. Fifty eight women were tested positive for HPV18. Of these 58, 39 were positive in all three samples, 5 in two and 14 in one. In the 60 women with HPV16 infection, median copy number was 7.7 in their first qPCR positive sample and 7.8 in their last positive sample. In the 58 women with HPV18 infection, median copy number was 2.3 in their first qPCR positive sample and 2.4 in their last positive sample. Viral load appeared to increase and decrease during follow up. Among 43 women with HPV16 infection and among 35 women with HPV18 infection, maximum viral load observed during follow up was found to be increased than that detected in the first qPCR positive sample and it was greater than that detected in the last qPCR positive sample in 37 of these women with HPV-16 infection and 32 with HPV18 infection.

Increase viral load is associated with an increase risk of acquiring an incident cervical cytological abnormality: There was no significant difference in the maximum HPV16 viral load or the maximum HPV18 viral load between women who subsequently acquired an incident cytological abnormality and those who did not. However, when viral load was modelled as a log10-transformed continuous covariate, controlling for whether or not a woman had ever tested positive for the relevant type using qPCR, it was observed that a 10 fold increase in HPV viral load was associated with a significantly increased risk of acquiring an incident cervical cytopathological abnormality in women with HPV16 or HPv18 infection.

 

Conclusions: Thus, the study concludes that while large relative increase in the viral load was associated with an increase risk of cytological abnormality, a single measurement of viral load done at an intermediate point during the natural history of HPV infection, does not reliably predict the risk of acquiring cervical neoplasia.

 

Source: Constandinou-Williams C, Collins SI, Roberts S, Young LS, Woodman CB, Murray

PG. Is human papillomavirus viral load a clinically useful predictive marker? A

longitudinal study. Cancer Epidemiol Biomarkers Prev. 2010 Mar;19(3):832-7.

Adenovirus vector vaccination induces expansion of memory CD4 T cells with a mucosal homing phenotype that are readily susceptible to HIV-1

A recently conducted phase 2B HIV vaccine trial in high risk HIV-negative volunteers (the STEP study) using an adenovirus vaccine (Ad5 vector expressing Gag, Pol and Nef) was ineffective. In that study, individuals were divided into two groups on the basis of their Ad5 antibody titers. Besides being ineffective, individuals who received this vaccine were more susceptible to HIV-1 infection in comparison to the placebo. This susceptibility was more significant in the group of vaccinees with high Ad5 antibody titers. The reason for this is not clear.

Natural Ad5 infection occurs via gut or nasopharynx and the virus replicates in the epithelial cells of mucosal tissues, inducing mucosal immunity. Thus, the authors of this study hypothesized that vaccination of individuals immune to Ad5 with adenovirus vector would activate and expand T cells expressing a mucosal homing phenotype, and these cells would migrate to the gut mucosa, increasing the number of permissive HIV-1 target cells and the risk of infection.

Citation: Benlahrech A, Harris J, Meiser A, Papagatsias T, Hornig J, Hayes P,

Lieber A, Athanasopoulos T, Bachy V, Csomor E, Daniels R, Fisher K, Gotch F, 

Seymour L, Logan K, Barbagallo R, Klavinskis L, Dickson G, Patterson S. 

Adenovirus vector vaccination induces expansion of memory CD4 T cells with 

a mucosal homing phenotype that are readily susceptible to HIV-1. 

Proc Natl Acad Sci U S A. 2009 Nov 24;106(47):19940-5.

 

RESULTS:

1. Adenovirus specific cytokine responses do not correlate with Ad5 antibody titers: Ad5 and Ad11 antibody titers were measured in 20 healthy donors. IFN-gamma ELSIPOT was performed on PBMCs stimulated with Ad5 or Ad11 from 15 of these donors. 73% and 66.7% of individuals showed responses against Ad5 and Ad11, respectively. When Ad5 and Ad11 IFN-gamma responses were plotted against Ad5 antibody titers, no significant correlation was observed in between the Ad5 or Ad11 IFN-gamma ELISPOT responses and Ad5 antibody titer. Furthermore, the investigators pulsed dendritic cells from the donor with Ad5, Ad11, tetanus toxoid, heat inactivated influenza or Staphylococcus enterotoxin B (SEB) and measured cytokines in lymphocytes. It was observed that majority of IFN-gamma responses against Ad5 and Ad11 were mediated by CD8 T cells. CD8 cells produced more TNF-alpha in response to stimulation with Ad5 or Ad11 in comparison to CD4 T cells. There was no or very little IL-12 production in response to either Ad5 or Ad11 by any cell. The authors then assessed the functional quality of the responding Ad5 and Ad11-specific CD4 T cells. There was no difference between Ad5 and Ad11 responding CD4 cells in terms of cytokine profile as both groups of cells predominantly produced TNF-alpha alone or in combination with IFN-gamma or IL-2.
   
2. Adenovirus induced T cell proliferation correlates with Ad5 antibody titers: The investigators next sought to determine the ability of Ad vectors to induce T cell expansion ex vivo. For this, DCs were pulsed with Ad5, Ad11, tetanus toxoid, heat inactivated influenza or Staphylococcus enterotoxin B (SEB) and co-cultured with autologous CFSE-stained T cells. It was observed that majority of proliferationg T cells (CSFR low) in response to Ad5 or Ad11 stimulation were CD4 T cells. No significant difference in CD4 or CD8 cells was observed in response to tetanus toxoid or influenza. The authors also observed a positive correlation between Ad5- or Ad11- induced CD4 T cell proliferation and Ad5 serostatus.
   
3. Alpha4beta7 expression by expanded adenovirus specific memory CD4 T cells: Since, migration of lymphocytes to gastrointestinal tissues is dependent on the expression of a heterodimer composed of alpha4 and beta7 integrins, the authors measured expression of this molecule on expanded CSFE-low (dividing) CD4 T cells co-cultured with Ad5-, Ad11-, tetanus toxoid-, heat inactivated influenza- or Staphylococcus enterotoxin B (SEB)-pulsed DCs for five days. Majority of the expanded Ad5-specific CD4 T cells expressed high levels of alpha4beta7 in comparison to CSFE high (undivided) CD4 cells.
   
4. Adenovirus induced alpha4beta7 increases correlate with Ad5 titers: The authors next determined the fold increase in the alpha4beta7 expression by stimulation of T cells with Ad5, Ad11, tetanus toxoid, influenza or SEB-pulsed DCs in relation to the background expression levels of CD4 T cells cultured with unpulsed DCs. A positive correlation between the fold increase in total CD4 T cells expressing alpha4beta7 in response to Ad5 or Ad11 stimulation was observed. The authors also found a positive correlation between the fold increase in total CD4 T cells expressing alpha4beta7 and Ad5 antibody titers.
   
5. CCR9 and CCR5 up-regulation in response to Ad5 and Ad11 challenge: As chemokine receptor CCR9 also helps in the migration of lymphocytes to mucosal tissues, the authors next studied the level of CCR9 expression by activated adenoviral-specific memory CD4 T cells in four donors. They observed significantly increased levels of surface CCR9 in cells expanded by stimulation with first and second generation Ad5 and Ad11 vectors in comparison to CSFE high cells. Similar to CCR9, CCR5, which functions as a co-receptor for HIV-1 entry is up-regulated by T cells with effector or memory phenotype and Th1 cells. The authors thus, investigated the expression of CCR5 on expanded adenovirus specific memory T cells. CCR5 was found to be up-regulated on CSFE low CD4 T cells when co-cultured with Ad5 or Ad11 pulsed DCs in comparison to CSFE high undivided T cells.
   
6. Re-stimulated adenovirus specific CD4 T cells are more permissive to HIV-1 infection: To determine the significance of increased expression of CCR5 on expanded adenovirus specific memory CD4 T cells, the authors stimulated DCs from these donors with first or second generation Ad5 or Ad11 and co-cultured them with lymphocytes. They were then infected with the R5 virus, HIV-1. The authors observed that intracellular staining for HIV-1 p24 was significantly higher in the proliferating CFSE-low CD4 T cells in comparison to un-dividing CFSE-high cells.
   

Oral Immunization with Attenuated Salmonella enterica serovar Typhimurium Encoding Cryptosporidium parvum Cp23 and Cp40 Antigens Induces a Specific Immune Response in Mice

Source of this paper is: Benitez AJ, McNair N, Mead JR. Clinical and vaccine immunology. 2009. 16(9):1272-1278.

The live oral Salmonella vaccines have been successfully used in delivering heterologous antigens and in generating a mucosal immune response against a number of organisms including bacteria, viruses and parasites. Among the parasitic species Toxoplasma gondii and Eimeria tenella are two good eaxmples. The advantages of attenuated Salmonella vaccines include:

• They induce both cell-mediated and humoral responses
  
• They elicit both systemic and local response
  
• They are easy to administer
  
• They are affordable
  

In this paper from the laboratory of Dr. Jan R Mead, Emory University School of Medicine, Atlanta, Georgia, the authors assessed the use of an attenuated strain of Salmonella carrying specific C. parvum antigens as a vaccine receptor and the protection that it offers against Cryptosporidium parvum infection.

Cryptosporidium parvum is an obligate intracellular protozoan parasite that causes gastrointestinal symptoms when ingested by humans. Infection with C. parvum can be severe and even life threatening in immuno-compromised individuals. Treatment is available but it could it could be less efficient in immuno-deficient individuals, thus, development of a vaccine that is capable of at least inducing partial immunity in such individuals would be beneficial.

The experiments performed and results obtained are:

1. Expression of Cp23 and Cp40 S. Typhimurium constructs: Cp23 and Cp40 genes from C. parvum were PCR amplified and sub-cloned into suitable expression vectors. The resulting constructs were transformed into Salmonella vaccine strain SL3261 (this strain contains a mutation in the aromatic amino acid biosynthetic pathway and has been previously reported to be the safest vaccine strain) and the expression of proteins was tested by SDS-PAGE and Western Blotting using mouse C. parvum serum. Both proteins were stably expressed in both aerobic and anaerobic conditions and in vaccine strain SL3261 and remained soluble.
   
2. Plasmid stability and Salmonella persistence in vivo: SL3261 containing the Cp23 protein was passaged in antibiotic free medium for a period of nearly five days. The plasmid was stably maintained when plated on to medium with or without antibiotics. Furthermore, three groups of mice were inoculated orally with SL3261 and euthanized at days 3, 10 and 25 post-immunization and bacterial counts per time point were performed on homogenates of spleen, liver and intestines. These counts showed that the recombinant construct grew and persisted in the reticulo-endothelial system.
   
3. Immunogenicity of Salmonella derived Cp23 and Cp40 antigens: Mice were immunized with SL3261 without Cp23 or Cp40 and with SL3261 with Cp23 and SL3261 with Cp40. Cp23 and Cp40 antibody production was determined in vaccinated mice at seven weeks after immunization. Specific serum Ig G levels were enhanced in mice immunized with Sl3261/Cp23 or SL3261/Cp40 in comparison to mice immunized with the control. Furthermore, the oral immunization with SL3261/Cp23 or SL3261/Cp40 induced only an IgG1 antibody response and no IgG2a was detected. These findings suggested that in response to immunization with S. Typhimurium encoding C. parvum Cp23 and Cp40 antigens, a Th2 type response is generated. It was also observed that a prior immunization with a recombinant DNA vaccine resulted in significant increase in titres of IgG specific anti-Cp23 and anti-Cp40 antibody response.
   
4. In vitro detection of neutralizing antibodies: To check whether antibodies from orally immunized mice can inhibit parasite from infecting host cells, sera from immunized mice were incubated with sporozoites and they were then allowed to infect HCT-8 cells. It was observed that sera from mice immunized with either Cp23 or Cp40 were able to reduce infection by sporozoites by 30%.
   

Conclusions:

The study demonstrates the immunogenicity of a single oral immunization with Salmonella vaccine strain SL3261 that expresses recombinant C. parvum antigens (Cp23 and Cp40) in mice. The study also shows the stability and safety of the recombinant Salmonella vector and a partial inhibition of infection by sporozoites incubated with immune sera. Although with some optimizations are still required, the use of Salmonella vaccine vector to deliver C. parvum antigens appears to be a feasible way to elicit humoral and mucosal immune response in the immunized host,

A review on Microfluidic systems for detection of pathogens

Being associated with molecular diagnostics of infectious agents for past so many years, I can easily identify with the need to develop faster, portable and more accurate methods for diagnosis of pathogenic microorganisms. For the last few years, much advancement has been made in this regard. This blog is on a very interesting review article from the laboratory of Dr. Peter Ertl, Viena, Austria on microfluidic systems for pathogen sensing.

Microfuidics is explained in Wikipedia as one that deals with the behavior, precise control, and manipulation of fluids that are geometrically constrained to a small typically sub-millimeter scale. It is a multidisciplinary field intersecting engineering, physics, chemistry, biotechnology, and microtechnology. It involves design of systems in which small volumes of fluid are used. It is used in the development of inkjet printheads, lab-on-a-chip technology, micro-propulsion and miceo-thermal technology.

The standard methods which are used for pathogen detection include pathogen culture, enzyme immuno assays and polymerase chain reaction. All these methods take 2-4 days and generally require to be performed in centralized labs. As most centralized labs are limited to larger cities, there is a need to develop near patient testing which is commonly referred to as point of care (POC) testing. Therefore robust and portable diagnostic devices capable of rapidly providing information on pathogens will also help reduce mortality rates and hospitalization in case of infectious pathogens. In the last two decades a variety of different biosensors have been developed. Still, miniaturized, low cost or disposable biosensors capable of rapid detection and accurate identification of a wide range of pathogens are required. Thus, efforts have been made to minimize the time span between sampling and results. The results of these efforts are miniature devices that do not depend upon special infrastructure and sample preparation procedures. The area of miniaturized or microfluidic analysis systems, which are also called "micro total analysis system" or "lab-on-a-chip (LOC) system".

The major advantages of the microfluidic systems are:

• To conduct measurements from small volumes of complex fluids with efficiency and speed.
  
• No need for a skilled operator
  
• Reduced consumption of reagents due to small size
  
• Ability to integrate separation and monitoring techniques within a single device.
  
• Portable LOC devices are able to provide information in even in most remote settings.
  

In this review the recent progress within microfluidic based pathogen detection is presented. A schematic overview of microfluidic based pathogen sensing is presented in this flow chart.

Source: Mairhofer J, Roppert K, Ertl P. Microfluidic systems for pathogen sensing: A Review. Sensors 2009, 9, 4804-4823.

Host cell damage induced by porcine ETEC

Enterotoxigenic Escherichia coli modulates host intestinal cell membrane asymmetry and metabolic activity

Dr. Philip Hardwidge of university of Kansas Medical Center has been working on several types of E. coli, that cause diarrhea and malnutrition in humans and livestock, for quite some time. In this particular article published in 2008 in infection and immunity, he and his team tries to find out the extent to which enterotoxigenic E. coli (ETEC) damages host cells.

ETEC is a common cause of traveler’s and postweaning diarrhea in humans and swine, respectively. ETEC releases several enterotoxins, which lead to water and electrolyte loss from the intestine. These toxins include heat labile enterotoxin (LT), the heat stable enterotoxin (ST) and the enteroaggregative E. coli heat stable enterotoxin 1. It has been earlier observed that several bacteria e.g., Salmonella induce apoptosis of host cells to promote their survival and dissemination. The same has been shown by several serotypes of E. coli and toxins; however, whether ETEC also induces apoptosis of host cell is not known.

Keeping in view of all this information, the work was conceptualized with following objectives:
1. to quantify the ability of ETEC and LT in mediating damage of porcine intestinal epithelial cells,
2. to clarify the role of LT in these processes, and
3. to measure any resultant increases in ETEC adherence.
A summary of results obtained is as follows:

ETEC induces an increase in phosphatidylserine (PS) exposure:
Phosphatidylserine (abbreviated Ptd-L-Ser or PS) is a phospholipid component, usually kept on the inner-leaflet (the cytosolic side) of cell membranes by an enzyme called flippase. When a cell undergoes apoptotic cell death phosphatidylserine is no longer restricted to the cytosolic part of the membrane, but becomes exposed on the surface of the cell.

IPEC-J2 cells were infected with wild type ETEC, a mutant strain deficient in LT expression and a mutant complemented by plasmid based eltAB expression. PS exposure was determined by annexin V staining and cell death was determined by PI staining.
Infection with wt significantly increased the PS exposure while insignificantly increased the cell death compared to that of uninfected cells. Infection with both the mutants also increased the PS exposure to same exten; it also increased cell death. Administration of LT only, modestly increased the PS exposure. Infection with a strain (G58-1) that lacks any known plasmids, produced no enterotoxins and is considered to be a commensal, did not significantly change the PS exposure. Further, the authors incubated host cells with outer membrane vesicles (OMVs) purified from ETEC possessing or lacking LT. It was observed that incubation with OMVs significantly increased the PS exposure irrespective of the presence of LT. however, no significant increase in cell death was observed due to purified OMVs or LTs.

All these results indicate that ETEC causes alterations in the membrane structure of intestinal epithelial cells. These alterations are the ones which are commonly associated with apoptosis. The results also indicate that LT is unlikely the primary determinant of this activity.

ETEC inhibits host calcein-AM degradation
To determine if ETEC also inhibits or alters metabolic activities of the host cells, the investigators infected host cells with different ETEC strains (i.e., wild type, mutants and G58-1) and then incubated the infected cells with calcein-AM. Calcein-AM is a non-fluorescent lipophillic ester that penetrates the cell membrane. Inside the cell, cytosolic esterases act on this calcein-AM and generate calcein, which is a fluorescent non-membrane permeable molecule. Thus, quantification of calcein fluorescence after uptake of calcein-AM provides a convenient way to assess cell viability and metabolic activity. It was observed that the conversion of calcein-AM in ETEC infected cells was significantly inhibited in comparison uninfected cells.
The authors also observed that although a large number of cells failed to stain with PI (that is they were live) they had very limited fluorescence. The authors gated these cells to quantify percent of calcein fluorescence negative cells among only live cells. They observed that 4% of live, uninfected cells had negative calcein fluorescence, while 62% of infected cells had negative calcein fluorescence. Infection with both the mutant strains also significantly inhibited calcein fluorescence. To determine if host calcein fluorescence would recover following removal of ETEC, the investigators treated the host cells with gentamycin and then quantified calcein fluorescence as a function of time after gentamycin treatment and they observed that percentage of calcein negative cells decreased from 62% to 14.3% over 1hr following the addition of gentamycin. The results of these experiments suggested that the metabolic activity of the host recovers rapidly after removal of ETEC.
All together, these data indicate that porcine ETEC profoundly alters the membrane asymmetry and metabolic activity of infected host cells.

ETEC does not induce host DNA double-strand breaks:
Double stranded DNA breaks are known to occur in late stages of apoptosis. To determine whether ETEC also causes such breaks, the investigators employed TUNEL assay. No significant difference in TUNEL staining was observed as a result of ETEC infection. These data indicate that while ETEC may induce changes to the host cell which are associated with early stages of apoptosis, e.g., alteration in membrane structure, subsequent signal transduction events mat be inhibited at a later time point in the pathway.

Diversity in porcine ETEC propensity to induce host cell damage:
The authors next used 11 other, different ETEC strains to infect host cells. The results of PS exposure were:
--Three strains induced PS exposure in more than 20% of cells
--Two strains induced PS exposure in 10 to 15% of cells
--Remaining six strains did not induce significant PS exposure
The results of calcein fluorescence experiments were
--Four strains reduced host calcein fluorescence levels equivalent to that observed with the earlier wt strain.
--Three strains has an intermediate phenotype.
--Remaining four strains did not induced any significant decrease in calcein fluorescence.

Furthermore, the strains that induced PS exposure and reduced calcein fluorescence, except one, are known to display either K88a or F41 fimbrae and efficiently bind to the cell lines used in these experiments.
The strains that failed to induce changes in PS exposure or host calcein fluorescence levels are known to display F18 or unknown fimbria phenotypes yet still possess various enterotoxins. These observations lead to the idea that the ETEC enterotoxins are not significant in mediating early apoptotic changes and an important step in causing apoptosis is adherence to host cells.

Host cell damage increase ETEC adherence:

The authors then tested whether caspases are activated in response to ETEC infection, as caspases play important role in apoptosis of cells. They found that both the wild type and mutant strains activated host caspase 3, whereas infection with LT or G58-1 did not activated the caspase 3.
The authors next hypothesized that inducing host cell apoptosis would increase host cell adherence. To test this, cells were treated with camptothecin (promotes apoptosis) and then with ETEC and subsequent adherence was determined. It was observed that pretreatment of host cell with camptothecin significantly increase adherence of ETEC to host cells. Importantly, this increase was observed in both wild type and mutant strains. These results suggested that increase in adherence is independent of the LT expression. To further determine if increase in adherence was dependent upon the activation of host caspases, the authors co-treated the host cells with camptothecin and Ac-DEVD-CHO, an inhibitor of caspase3 dependent pathways. It was observed that Ac-DEVD-CHO caused the adherence to decrease to basal levels.
Taken together all these data indicate that inducing apoptosis in host cells is sufficient to promote subsequent ETEC adherence and that this phenomena require the activity of caspase 3 and/or the downstream effectors of this enzyme.
Furthermore, to assess whether any factor is secreted by apoptic cells in response to chemical induction of apoptosis that might be sensed by ETEC and helps to promote adherence, the authors conducted an experiment. They treated host cells with camptothecin and then treated one set of cells with Ac-DEVD-CHO. They then filtered the supernatant from these cells and added it to culture of ETEC. After 24hrs of incubation, these ETEC inocula were used to infect host cells. It was observed that presence of both camptothecin and Ac-DEVD-CHO abolished the adherence promoting ability of ETEc. Thus, it was possible that a factor released from host cells after activation of caspase 3 apoptic pathways may play a role in regulation of gene expression of pathways associated with ETEC adherence.

Cathepsin Cs and their role in survival of Toxoplasma gondii

Cathepsins Cs are key for the intracellular survival of the protozoan parasite, Toxoplasma gondii

Toxoplasma gondii is an opportunistic pathogen that causes serious disease in AIDS patients, recipient of solid organ transplants and in newborns who acquire the infection in utero. The life cycle of the parasite consists of two phases, i.e., asexual and sexual. The sexual phase takes place only in the members of felidae family (domestic and wild cats). The asexual phase can take place in any warm blooded animal. Infection in humans is acquired by ingesting oocysts. Inside the body sporozoites are released that invade macrophage of the intestines. These sporozoites are differentiated into tachyzoites which are motile and are distributed to other parts of the body through blood. In an immunocompetent host, the actively dividing tachyzoites differentiate into metabolically inactive bradyzoites, leading to life-long latent infection. Tachyzoites can invade any nuclear cell and survive intracellularly in a specialized parasitophorous vacuole in which the tachyzoites enter into bradyzoite stage.
Cysteine proteinases of the parasite play key roles in pathogenesis of the parasite and help in host invasion, parasite differentiation and intracellular survival. Cathepsin C also known as DPPI is one such cysteine protease that belongs to the papain family of cysteine proteases. The other proteases included in this family are cathepsin B, L, K, H and S. In contrast to these cathepsins, which have endopeptidase activity, cathepsin C has primarily exopeptidase activity. The authors of this paper have earlier characterized T. gondii cathepsin B and cathepsin L proteases. In the present work the authors show that T. gondii cathepsin Cs play important roles in tachyzoite growth and differentiation and may be used as drug targets in future.
This interesting paper is from the lab of Dr. Sharon L Reed, Department of Pathology and Medicine, University of California, San Diego, USA.

Expression and characterization of cDNAs encoding T. gondii cathepsins C:
The authors identified and cloned three cathepsin C genes from T. gondii.

Expression of cathepsins in tachyzoites and bradyzoites: The authors next studied the relative expression of different cathepsins (B, L, C1, C2 and C3) in tachyzoites using real time qPCR. Total RNA from in vitro cultured tachyzoites of T. gondii was isolates and transcribed into single stranded cDNA. Primers were designed to sequences of beta-tubulin, a tachzoite specific gene (SAG1), and cathepsins C1, C2, C3, B and L. The highest level of expression was shown by TgCPC1. The mRNA of TgCPC1 was 64% of the total TgCP mRNA. TgCPB was 21% and TgCPL was 7% of total CP RNA in tachyzoites. TgCPC3 mRNA was below the level of detection. The authors next performed a comparative real time PCR on the cDNA isolated from in vitro cultures tachyzoites and mature, encysted bradyzoites isolated from brains of chronically infected mice. TgCPC1, TgCPC2 and TgCPB were detected from tachyzoites but not from bradyzoites whereas TgCPL was detected in both.
These data suggest that the cathepsin genes are stage specifically down regulated.

Purification and catalytic properties of T. gondii cathepsin C:
The authors expressed cathepsin C1 and C2 in E. coli and also purified native enzymes from tachyzoites. These recombinant cathepsins and native cathepsins were run on a SDS PAGE and reacted with anti-CPC1 antibodies. Attempts to refold recombinant TgCPC1 and TgCPC2 as well as expression in Pichia were unsuccessful.
Using native enzymes, it was observed that resulting cathepsin C was 80% pure and reacted with TgCPC1 specific antibodies by Western blot analysis. No cross reaction with TgCPC2 antibody was observed. Furthermore, five synthetic dipeptide substrates were used to characterize the proteanse activity of purified TgCPC1 fractions.

Inhibitor profile of T. gondii cathepsin C and Inhibition of multiplication by cathepsin C inhibitor:
Host cells (fibroblasts) were infected with tachyzoites for 2hr and then after washing away the free parasites, they were treated with cathepsin C inhibitor. Cultures were incubated for 24hrs, fixed and stained with acridine orange and the number of parasites per vacuole was counted by fluorescence microscope. It was observed that cathepsin C inhibitor significantly reduced intracellular replication of inhibitor treated tachyzoites. The authors also tried to compared the morphology of inhibitor treated and control parasites by fluorescent and electron micrsoscopy. No abnormal morphology of parasites or fibroblasts was detected.

TgCPC1 knock-out mutant:

The authors then generated a TgCPC1 knock out through gene disruption and analyzed the phenotype of the resulting mutants. They compared the ability of both wild type and mutant parasites to invade and replicate in host cells. After twenty-four of invasion of the host cell, there were no significant differences in the number of cells infected or the average number of parasites per vacuole between the mutant TgCPC1 mutants and the wild-type parasite.


Localization of TgCPC:
TgCPC1 and 2 were found in dense granules in tachyzoites but not in rhoptries or micronemes.

TgCPC degrades proteins in the parasitophorous vacuoles of T. gondii: The results of these experiments suggest that cathepsin Cs are either directly or indirectly required to degrade proteins or oligopeptides in the vacolar space.

Phoneix Identification of ESBL producers

Source: Fisher MA, Stamper PD, Hujer KM, Love Z, Croft A, Cohen S, Bonomo RA, Carroll KC, Petti CA. Performance of the Phoenix bacterial identification system compared with disc diffusion methods for identifying extended-spectrum beta-lactamase, AmpC and KPC producers. J Med Microbiol. 2009 Jun;58(Pt 6):774-8.

Beta-lactamases are enzymes produced by some bacteria and are responsible for their resistance to beta-lactam antibiotics e.g., pencillins, cephamycins and carbapenems. Extended-spectrum beta-lactamases (ESBLs), AmpC and KPC beta-lactamases are some of the common beta-lactamases produced by the members of Enterobacteriaceae. Accurate detection of beta-lactam producers is critical for decision making in therapeutic and infection control. There are many methods of ESBL detection, some of them are automated while others are manual. Recently, Phoenix Automated Microbiology System has been designed by BD diagnostics.

In the present study, the authors (1) compared the disc diffusion (DD) method of Clinical and Laboratory Standard Institute (CLSI) with Phoenix method for identifying ESBL+ isolates of K. pneumoniae and E.coli, (2) sssessed the value of an extended phenotypic method [cefpodoxime and aztreonam±clavulanic acid (CA)] for enhancing ESBL detection, (3) evaluated boronic acid (BA) with cefotetan or ceftazidime±CA to identify AmpC producers that could mimic or mask ESBL production, and (4) assessed these phenotypic tests with KPC-positive clinical isolates.

Conclusions:

Of 100 isolates identified as ESBL- by Phoenix system, 4 were false negative as determined by DD testing. OF the 10 isolates identified as ESBL+ by Phoenix system, 46 were false positive.
Half of the KPC+ isolates were reported as carpenem –susceptible by Phoenix system.

TLR expression and responsiveness of intestinal DC subsets

Hypo-responsiveness of intestinal dendritic cells to TLR stimulation is limited to TLR4

This very interesting paper is from the lab of Dr. Linda Klavinskis, Department of Immunology, King’s College, London.

As the intestine encounters a plethora of antigens, some of which are harmful while others are harmless, immune mechanisms have evolved to maintain immunological tolerance against harmless antigens to avoid chronic inflammation and to elicit protective responses against harmful antigens. One of the major unanswered questions in mucosal immunology is how this delicate balance is maintained in the gut.
The current literature suggests that dendritic cells (DCs) play important roles in immune regulation. It is assumed that DCs are activated on detection of microbe associated molecular patterns (MAMPs) by pathogen recognition receptors (PRRs) on DCs. Of the various PRRs, TLRs are mostly widely understood and they recognize a variety of MAMPs. However, very little is known about the capacity of intestinal DCs to detect and respond to MAMP or TLR-agonists.

Before talking about this paper, let me summarize the current knowledge on this topic. It has been observed that CD11c+ lamina propria (LP) cells (which include DCs also) were not found to be expressing TLR4 mRNA and steady state and were also found not to respond to the LPS stimulation in vitro. Similarly, mouse CD103+ mesenteric lymph nodes (MLN) DCs, which are thought to be derived from intestine, showed lower levels of TLR2 and TLR4 mRNA than CD103- MLN DCs (thought to be blood derived cells). All these studies suggest that down-regulation of TLRs on DCs could be responsible for immune tolerance to harmless antigens. However, a recent study showed that murine CD11chighCD11bhigh LP DCs express TLR5 and respond to flagellin stimulation. The authors of the current paper have also previously shown that a subset of rat intestinal lymph DCs respond to TLR7/8 agonists stimulation.

All these studies indicate that intestinal DCs might not be unresponsive to all TLR agonists and that they might express some TLRs.

The capacity of intestinal DCs to express PRRs and respond to MAMPs is a key question and its answer can aid in design of oral vaccines and provide indication of pathways that lead to inflammation. Thus, the authors in this paper sought to determine the pattern of TLR expression and functionality of intestinal DCs. The problem with the study of intestinal DCs is that the isolation procedure may itself induce phenotypic and functional changes in DCs. To overcome this problem, the authors in this paper collected lymph-born DCs from the thoracic duct lymph of mesenteric lymphadenectomized (MLNX) rats. All these intestinal lymph-borne DCs (iLDC) express high levels of CD103 and MHCII. They represent a heterogeneous population distinguished by the expression of CD172a and CD11b.

The authors showed that iLDCs were unresponsive to TLR4 agonist LPS but were able to up-regulate activation markers up on stimulation through other TLRs.


Steady State Repertoire of TLRs expressed by intestinal DCs

Experiment: iLDCs were collected from the lymph of MLNX rats. They were enriched using anti-CD-103 magnetic beads and sorted by FACS as large CD103+ MHC class II+ cells. There were three subsets of DCs. They were CD172a-, CD172a+ CD11b- and CD172a+CD11b+. On these cells the expression of mRNA of TLR1 to 11 and MD2 (a co-receptor for TLR4) was determined by qPCR and the expression levels were compared with those of Fl3l generated rat BMDCs.

Results:
1. Rat iLDC expressed mRNAs of all TLRs (1-11) except TLR7.
2. No significant difference in level of expression of TLRs 5, 6, 10, 11 and MD2 was observed in any subsets of DCs or in BMDCs.
3. The level of TLR8 mRNA was comparable in CD172a+ DCs and BMDCs.
4. The level of TLR3 mRNA expression was significantly higher in CD172a- in comparison to CD172a+ cells.
5. TLR9 was similarly expressed by all three subsets of DCs.
6. TLR4 was significantly low in all three cells than in BMDCs.
7. TLR2 levels were high on all three DCs and comparable to BMDCs.

Conclusions:
These data suggest that iLDCs express transcripts of all TLRs (except TLR7) in steady state. However, the level of expression varies in different populations of DCs.

Can intestinal DCs be activated by TLR agonists in vitro?
Experiments: Enriched iLDCs were cultured for 18hrs in the presence or absence of Pam3Cys (TLR-2 agonist), Poly (I:C) (agonist signaling through TLR3, although it can activate other cellular receptors), LPS (TLR4 agonist), flagellin (TLR5 agonist), R-848 (TLR7/8 agonist), and CpG (TLR9 agonist). The levels of activation markers CD25 and CD86 were assessed by flow cytometry.

Results: With the exception of TLR4 agonist LPS, a significant increase in percentage of cells expressing CD25 or CD86 was observed in case of culture with all other TLR ligands.

Conclusions: These data suggest that despite continuous exposure to intestinal commensal bacteria, intestinal DCs respond to TLR agonists.

Can highly pure intestinal DCs be activated by TLR agonists in vitro?
Experiments: CD103+ and MHC II+ iLDCs were sorted by FACS and cultured for 18hrs in the presence or absence of a range of TLR2 agnosits (Pam3Cys, Pam2Cys, and heat-inactivated spores of B. subtilis). The levels of activation markers CD25 and CD86 were assessed by flow cytometry. Similar experiments were repeated with TLR9 agonist CpG. Further more, the production of cytokines in response to stimulation with TLR liagnds was determined. The three populations of DCs were sorted by FACS and incubated with TLR ligands.

Results:
1. A significant increase in percentage of cells expressing CD25 or CD86 was observed in case of culture with all TLR2 and TLR9 ligands.
2. The authors also found that DCs cultured with Pam3cys, Pam2Cys, B. subtilis spores or CpG responded with enhanced secretion of IL-6 and IL-12p40 in comparison to DCs cultured alone.
3. DCs incubated with LPS did not show any enhanced secretion.
4. All three subsets of DCs secreted IL-6 and IL-12p4o inresponse to TLR2 and TLR9 ligands but not to TLR4 ligand.
5. Of the three subsets, CD172a-, but not the CD172a+CD11b- or the CD172a+CD11b+ iLDCs, secreted TNF-alpha when cultured with Pam3Cys or CpG, but not with LPS.

Intra-intestinal injection of B. subtilis spores induces a significant increase in proportion of activated DCs migrating out of intestines
Experiments: MLNX rats were injected intra-intestinally with heat inactivated spores of B. subtilis or with PBS as control. After 24 hr, thoracic duct lymph cells were collected.

Results:
1. There was no change in the proportion of intestinal DCs migrating to lymph in two groups of animals.
2. A small but significant increase in the number of DCs bearing CD25 or CD86 was detected in lymph 24 hr after injection with spores in comparison to controls.
3. Same response was observed in all three DC subsets.

Conclusions: The intra-intestinal injection of a model non-invasive Gram-positive organism causes a significant increase in up-regulation of activation markers in DCs migrating from intestine to lymph.

Do these migrating DCs directly interact with intra-intestinally injected B. subtilis spores?
Experiments: MLNX rats were injected intra-intestinally with heat inactivated CFSE labeled spores of B. subtilis or with PBS as control. After 24 hr, thoracic duct lymph cells were collected. DCs were identified as CD103+ and MHCII + cells by flow and evaluated for green fluorescence (due to CFSE) and CD25 expression. CSFE+ and CSFE- cells were sorted by FACS and mRNA levels of cytokines were examined.
Results:
1. A marked increase in green fluorescence was observed in rats injected with FCSE labeled spores in comparison to controls.
2. There was preferential activation of spore bearing DCs as the fluorescence was exclusively limited to CD25+ iLDCs.
3. CSFE+ DCs showed a 3-5 fold increase in mRNA expression of IL-6 in comparison to controls.
4. No significant increase in mRNA levels of other cytokines (IL-10, TNF-alpha, IL-12p40, IL-12p35 or IL-23p19) was observed.
5. Also, when FACS sorted CD103+ MHC II+ cells from the lymph of spore injected mice were analyzed by fluorescent confocal microscopy, bright green colored spores clearly visible within the DCs were observed.

Conclusions: There is direct interaction of intestinal DCs with B. subtilis spores which leads to activation of DCs and these spores are internalized by DCs.

Mycobacterium tuberculosis PPD-induced immune biomarkers measurable in vitro following BCG vaccination of UK adolescents

Source: Smith SG, Lalor MK, Gorak-Stolinska P, Blitz R, Beveridge NE, Worth A, McShane H, Dockrell HM. Mycobacterium tuberculosis PPD-induced immune biomarkers
measurable in vitro following BCG vaccination of UK adolescents by multiplex bead
array and intracellular cytokine staining. BMC Immunol. 2010 Jul 7;11:35.

Mycobacterium tuberculosis PPD-induced immune biomarkers measurable in vitro following BCG vaccination of UK adolescents by multiplex bead array and intracellular cytokine staining

Clinical trails of BCG vaccination have previously shown variable efficacy against pulmonary tuberculosis in adults (0-80%) and high efficacy in children. BCG vaccination has demonstrated a protective efficacy of 77% against pulmonary tuberculosis when administered to UK adolescents. In this paper, the authors have used this setting to investigate the nature of immunity induced by BCG vaccination in a cohort of school children (12-15 years) in UK.
The Th-1-type immune response is most effective against TB. IFN-gamma which is an important part of Th-1-type immune response plays important role in protection against TB, as mice and human who are deficient in IFN-gamma signaling pathways are highly susceptible to TB. However, it has also been observed that patients with a latent TB infection and positive IFN-gamma release status can progress to active infection and IFN-gamma can also be detected in samples from patients with active disease. Thus other components of type-1 response e.g. TNF-alpha, IL-2 and IL-12 may also play important roles.
A critical role of Th-17 response involving IL-17 has also been suggested by some studies. CD4 cells play important role in TB immunity. Other cells that are also important include CD8 cells, natural killer T cells and gamma delta T cells. A protective biomarker profile thus defines the presence of all these cytokines and cells that secrete these cytokines and absence of certain biomarkers (e.g., IL-4 and IL-10) which may subvert the response to TB and allow bacterial infection to proceed.

Results:
Biomarkers detected by diluted whole blood assay and multiplex bead array analysis: Blood sample from 12 adolescents were taken before and after BCG vaccination. These blood samples were diluted with RPMI 1640 and cultured for six days in the presence or absence of Mtb purified protein derivative (PPD). Assay supernatants were collected and frozen and analyzed for biomarker content by multiplex bead array. Of the 20 biomarkers that were measured in this study, 10 were found to be raised in samples form adolescents taken after one month of vaccination. These included Th1 type cytokines, IFN-gamma and IL-2, proinflammatory cytokines and chemokines, TNF-alpha, IL-17, IL-1alpha, MIP1alpha, IL-6 and IP-10. GM-CSF and anti-inflammatory cytokine, IL-10 were also elevated. No significant increase was observed in G-CSF, IL-4 or IL-8. Some cytokines were undetectable in both pre and post vaccination samples. These were IL-12p70, IL-7, IL-15, IL-5, IL-1β and eotaxin. The authors further wanted to know which biomarkers were associated with IFN-gamma responses in these assays. They found that there was a correlation between the magnitude of IFN-gamma responses and those of other Th-1 or proinflammatory cytokines such as TNF-alpha, MIP-1alpha, IL-2, and IL-17. The authors also observed a strong correlation between IFN-gamma and GM-CSF and between IFN-gamma and IL-10. The authors then analyzed biomarker profile in children after 12 months of vaccination. It was observed that out of 10 biomarkers that were significantly increased in post 1 month vaccinated samples, 8 were also increased after 12 months of vaccination. IL-6 and IL-10 returned to levels comparable with pre-vaccinated samples.
Thus, the authors concluded that BCG vaccination results in a broad biomarker response measurable at one month in peripheral blood and detected following stimulation with Mtb PPD. The authors also observed that this biomarker response is maintained up to 12 months of vaccination.

Flow-cytometric analysis of cellular cytokine production
The authors isolated and preserved PBMCs from blood samples of 12 months post BCG vaccinated children. The recovered cells were stimulated for 24 hrs in the presence or absence of Mtb PPD and then stained with a panel of antibodies that recognize phenotype markers of lymphocytes and cytokines. First the authors applied gating on singlet and live cells and then excluded CD14 or CD19 expressing cells. After this, the single cytokine expression was examined on CD4 (CD3+ and CD4+), CD8 (CD3+ and CD8+) and NK (CD3- and CD56+) cells. The authors found that IFN-gamma was detected in all three set of cells. TNF-alpha was detected in CD4 cells and NK and it was very low in CD8 cells. Next the authors studied the expression of both IFN-gamma and TNF-alpha in these samples. They found that majority of cytokine producing cells were either producing IFN-gamma or TNF-alpha and very few were producing both.
Thus, from these studies the authors concluded that cytokine responses are less readily detected by flow cytometry compared to multiplex bead array analysis involving a six day culture.

Conclusions:
In the present study, the authors demonstrated Mtb PPD specific response after 1 month and 12 month of BCG vaccination. This response was quite broad in terms of cytokines released. These cytokines included Th-1 responses and pro-inflammatory cytokines and chemokines. When the PBMCs from 12 month vaccinated samples were analyzed by flow cytometry, CD4 and CD8 cells that can release more than one cytokines were not detected. The majority of these cells and also NK cells were single cytokine producers.

Future aspects:
IL-6, IL-8 and IP-10 were elevated in post vaccine samples on stimulation with Mtb PPD, however, they were also high in pre vaccine samples on stimulation with Mtb PPD. The reason for this is unknown. The authors hypothesized that there could be some antigen non- specific stimulation of macrophages and monocytes via the TLRs in response to undefined components of Mtb PPD protein cocktail.

IL-17 was found to be highly increased in samples from post vaccinated children. However, the precise nature of IL-17 detected remains unknown. Protein antigens in Mtb PPD that are processed and presented by MHC II molecules can lead to CD4+ Th17 response. The other possible source could be gamma delta T cells.

Filamentous influenza virus A infection predisposes mice to fatal septicemia following super-infection with Streptococcus pneumoniae serotype 3

This paper is from the lab of Dr. Paul C. Roberts, Department of immunology and microbiology, Wayne state university school of medicine, Michigan, USA.

Influenza A virus generally causes mild respiratory illness. Mortality in most cases is due to secondary bacterial super-infections. It has been observed that 44 to 57% of patients hospitalized with influenza virus, test positive for bacterial pneumonia. Streptococcus pneumoniae is one of the leading causes of community acquired pneumonia and also one of the leading causative agents in patients suffering from bacterial and viral co-infections.
Previous studies have shown that infection of mice with S. pneumoniae, who were previously infected with influenza virus showed increased colonization of the bacterium in the respiratory epithelium and decrease neutrophils recruitment. It has been proposed that influenza infection causes unmasking of Pneumococcal receptors by neuraminidase. The other idea is that influenza causes a depression in neutrophils function. However, majority of these previous studies were based on use of mouse adapted influenza A/PR8/34 virus for prior infection. This strain of mice is highly lethal to the mice and it does not mimic the typical mild course of disease progression observed in humans. Thus, in order to monitor the bacterial super-infection in a natural course of influenza infection in humans, the authors used the filamentous influenza A/Udorn/72 virus strain and S. pneumoniae serotype 3. This strain of influenza virus is highly filamentous and results in mild infection. Influenza A virus in general produces both spherical and filamentous virions. It has been shown that primary human isolates of influenza virus are predominantly filamentous. The S. pneumoniae serotype 3 was chosen as it is the most common serotype colonizing adult throats and is also associated with greater risks of death.

The authors found that filamentous influenza virus infection predisposes mice to fatal septicemia following super-infection with S. pneumoniae serotype 3. The authors also observed that high levels of granulocyte colony-stimulating factors (G-CSF) play a major role in synergistic exacerbation of disease.

The results of the experiments are described below:
1. Filamentous influenza A/Udorn/72 and S. pneumoniae serotype 3 both produced a mild, non-lethal infection in BALB/c mice when administered individually
a. Mice infected with A/Udorn/72 did not show overt clinical symptoms associated with influenza.
b. Minimum transient weight loss was observed.
c. Virus replication peaked at day three, followed by gradual clearance by day 4 – 6 and no virus detected by day 7.
d. At no time virus was isolated from brain, kidney or spleen.
e. Similarly, infection with S. pneumoniae serotype 3 resulted in a mild, self limiting infection.
f. Transient bacteremia was observed in30% on mice.
g. Systemic bacterial load declined rapidly and by day 7 bacteria had completely cleared from the body.

2. Type 3 pneumococcus administered during active influenza A/Udron/52 virus infection resulted in fatal septicemia: When mice infected with influenza A/Udorn/72 were super-infected with S. pneumoniae type 3, it was observed that
a. Super-infection at day 3 (peak of viral load) or day 5 (decline of viral load) caused bacteremia in 100% of mice and they died within three days.
b. Significantly higher bacterial loads in all organs including nasal tract and blood were observed in all mice.
c. There was delay in viral clearance from lungs; the viral loads were still detectable in two of the seven mice at day 8 of infection

3. The increase susceptibility to fatal septicemia is maintained well after virus clearance: Mice who were infected with influenza were allowed to recover and at day 14 they were inoculated with S. pneumoniae. This time point was chosen as there were no detectable virus beyond day 7, thus by day 14, mice had completely recovered from influenza.
a. Super-infection at day 14 resulted in 90% fatality rate.
b. Super-infected mice showed symptoms that were identical to those displayed by mice, which were super-infected with S. pneumoniae in presence of an active influenza infection.
c. Super-infected mice (at day 14) also showed higher bacterial loads in comparison to mice infected with pneumococcus alone.

4. Neutrophils dominate the lung inflammatory cell infiltrate in influenza virus- predisposed mice: Inflammatory cell infiltrates obtained by broncheo-alveolar lavage (BAL) were examined in different groups of mice. These groups were singly infected (with either Influenza or pneumococcus), super-infected (at different time points) and control (treated with PBS).
a. BAL fluid from singly infected and control mice largely consisted of alveolar macrophages (AM).
b. BALF from super-infected mice (at day 5) consisted mainly of neutrophils
c. Singly infected mice, however, showed lymphocyte infiltrations; 16% in pneumococcus and 34% in influenza.
d. Similar to mice that were super-infected at day 5, super-infection at day 14 also resulted in significant influx of neutrophils into the lungs.
e. On day 17 post-infection, BALF from singly infected and control mice consisted mostly of AMs, while BAL from super-infected mice at this time point (i.e., day 3 of post super-infection) consisted predominantly of neutrophils (65%).
f. At day 17 post-infection in mice singly infected with influenza, 13% cells were lymphocytes.

5. Histo-pathological analysis confirmed neutrophils accumulation in tissues:
a. Micro-scopical examination of the hemoatoxyin-eosin stained lung sections showed edema in four out of seven and neutrophilic infiltrates in seven out of the seven tissue samples.
b. Lung sections of singly infected mice were same as that of control mice with no pathological changes.

6. Cytokine and chemokine gene expression levels were selectively up-regulated in super-infected mice:
a. In contrast to singly infected and control mice, the levels of pro-inflammatory cytokines (IL-1beta, IL-6, TNF-alpha) were significantly up-regulated in mice which were super-infected at day 5 post influenza.
b. Similarly, gene expression of anti-inflammatory cytokine IL-10 was up-regulated in mice which were super-infected at day 5 post influenza.
c. The chemo-attractants, MIP-1beta, IP-10, MIP-2 and KC were also up-regulated mice which were super-infected at day 5 post influenza.
d. In the brains of super-infected (day 5) mice, the mRNA levels of IL-1beta, MIP-2 and KC were up-regulated.
e. In the spleen of super-infected (day 5) mice, the mRNA levels of IL-6, Il-10 and KC were up-regulated.
f. The mRNA levels of G-CSF were significantly up-regulated in the spleen, lungs and brains of super-infected (day 5) mice.
g. In the lungs of super-infected animals which were super-infected at day 14 post influenza, mRNA levels of IL-6, Il-10 and MIP-2 were up-regulated. However, IL-1beta, TNF-alpha and KC were not significantly up-regulated.
h. The spleens and brains of mice who were super-infected at day 5 post influenza, also showed elevated levels of expression of MIP-2, KC and G-CSF, although the levels of their expression was reduced a factor of 3.

7. Cytokine and chemokine levels were synergistically up-regulated in super-infected mice: Sera and BALF from mice were analyzed for the presence of cytokine and chemokines at day 5 and day 14 post infection.
a. Increase in production of IL-10, IL-6, TNF-alpha and KC was observed in both BAL and sera at both day 5 and day 14.
b. IL-1 beta was found to be elevated in BAL but not in sera of super-infected animals.
c. G-CSF levels were elevated in both sera and BAL from mice super-infected at day 5 and 14.

Conclusions:
 When mice are singly infected with either filamentous influenza A or with S. pneumoniae serotype 3, they are able to control the infection.
 An active influenza A infection is synergistically exacerbated by super-infecting S. pneumoniae leading to fatal disease.
 A prior influenza infection leads to the generation of a “predisposed state” and that active influenza infection is not essential for generation of this state. This predisposed state makes the mice susceptible for subsequent super-infection even after clearance of influenza.
 Influenza virus induced lymphocyte filtration may be responsible for the generation of predisposed state leading to exacerbated responses following super-infection.
 This predisposed state induced by a prior influenza infection can be maintained for at least 7 to 8 days following viral clearance and results in up-regulated secretion of the anti-inflammatory cytokine IL-10 together with several pro-inflammatory cytokines and especially G-CSF.

The PagN protein of Salmonella enterica serovar Typhimurium is an adhesin and invasin.

The PagN protein of Salmonella enterica serovar Typhimurium is an adhesin and invasin.
Lambert MA, Smith SG. The PagN protein of Salmonella enterica serovar
Typhimurium is an adhesin and invasin. BMC Microbiol. 2008 Sep 8;8:142.

Salmonella is a gram negative bacteria and is a member of the family Enterobacteriaceae. The nomenclature of Salmonella is complex. The earlier nomenclature was based on clinical considerations such as S. typhi-murium (mouse typhoid fever). When serological analysis was adopted, a Salmonella species was identified as a “group of related fermentation phage type” and each Salmonella serovar (serological variants) was classified as a separate species. Later, molecular studies led to the hypothesis that all Salmonella serovars form a single DNA hybridization group i.e., a single species and this species was composed of seven sub-species. The species name that was adopted was Salmonella enterica. Each of the sub-species contains various serovars. S. enterica serovar Typhimurium is one such species (belongs to sub-species 1 and serovar is Typhimurium).
It infects a wide range of animal hosts and caused self limiting gastroenteritis. The first step in pathogenesis is attachment of S. enterica serovar Typhimurium to the intestinal epithelial cells with the help of the fimbrae. The outer membrane proteins Rck and OmpD also mediate attachment. After attachment the subsequent process of uptake of Salmonella into mammalian cells is complex process. This process is coordinated by a series of proteins that are encoded within the SP-1 and SP-5 pathogenecity islands. The SP-1 locus encodes a type 3 secretion system (T3SS) that delivers effective protein into the host cells. The net result of ejecting of these effector proteins is entry of the organism into non-phagocytic cells.
Transcription of the virulence genes of Salmonella is regulated by the PhoPQ system. This system consists of PhoQ membrane bound sensor kinase and cytoplasmic DNA binding protein PhoP. When a specific external stimuli is present, this kinase phosphorylates the PhoP, which increases its binding affinity to the specific DNA targets. As a result, some genes are activated (Pags, PhoP activated genes) while others are suppressed (Prgs, PhoP repressed genes).
PagN is a PhoP activated genes. Its function is unknown, although it shows 54% homology with Hek and Tia adhesins/invasins of the pathogenic E. coli. In this exhaustive work done by Dr. MA Lambert and Stephen GJ Smith, Trinity College, Dublin, the authors show that when PagN was expressed in E. coli, it caused agglutination of erythrocytes and adhesion and invasion of mammalian cells. The authors also observed that S. Typhimurium mutants defective in PagN displayed reduced adhesion to and invasion of mammalian cells. Furthermore, it was observed that multi-copy expression of PagN can compensate for the loss of the SP-1 encoded T3SS.
The major results and methodologies used in the study are described as follows:
PagN is a heat sensitive hemagglutinin but not a autoagglutinin: The Hek protein of E. coli promotes hemagglutination. To determine whether PagN also promotes agglutination of erythrocytes, the authors performed a microtitre hemagglutination assay. For this assay, E. coli K12 strainXL-1 blue containing the PagN expression vector, pML1 or the empty vector control, pTrc99a, were incubated with human RBCs. It was observed that the presence of PagN expression vector caused the recombinant E. coli to agglutinate erythrocytes. As Hek is a heat resistant agglutinin, the authors next investigated whether PagN is also heat resistant. Unlike Hek, PagN showed a decrease in HA titres after heating thus showing that PagN is heat sensitive. The authors also observed that unlike Hek, PagN is not an autoagglutinin by a specteophotometric assay. The authors next wanted to know if PagN could support hemagglutination when expressed in multi-copy S. Typhimurium. Thus, plasmid pPagN2.3 was introduced into S. Typhimurium LT-2 and cultured in a medium that caused maximal induction of the PhoP dependent PagN promoter. To confirm the expression of PagN, the outer membrane extracts were analyzed by SDS-PAGE. To their surprise, the investigators observed that S. Typhimurium containing pPagN2.3 did not cause hemagglutination. Laboratory strains of E. coli such as K12 and B completely lack the O antigen of LPS. In contrast, the O antigen of S. Typhimurium is very large. Thus, the authors hypothesized that the large O antigen of S. Typhimurium may serve to mask the PagN and thus may limit access to its receptor. To investigate this, the authors used a S. Typhimurium strain CH133, a gal mutant, which lacks the O antigen component of LPS (rough strain). The authors transformed this strain with the plasmid pPagN2.3 and observed the agglutination of RBCs. These results suggested that LPS is capable of masking PagN and preventing functional receptor binding during hemagglutination.

PagN is an adhesin and invasin: To determine whether PagN could mediate adhesion to and invasion of eukaryotic epithelial cells, like its homologues Tia and Hek E. coli DH5 alpha harboring either control plasmid (pTrc99a) or PagN expression vector, pML-1, were induced with IPTG and incubated with eukaryotic cell lines CHO-K1. Approximately 60% of PagN-expressing bacteria were observed to invade the CHO-K1 cells. As invasion of epithelial cells by bacteria is often accomplished by inducing cytoskeletal rearrangements by manipulating polymerization of actin. Cytochalasin D is one of the cytochalasin that inhibits elongation of actin filaments at the fast growing end and thus it affects polymerization of actin. Thus, confluent CHO-K1 monolayers were pre-incubated with cytochalasin D for 30 minutes before infection with bacteria. When these cells were infected with bacteria, a 30 fold reduction in invasion was observed.

PagN contributes to Salmonella adhesion to and invasion of epithelial cells: A pagN mutant strain ML6 was constructed. It was observed that both wild type SL1344 and pagN mutant strain showed the same doubling time, thus removing PagN does not affect growth of S. Typhimurium. Also, the LPS profiles of both strains were also similar. The contribution of PagN to the invasion of CHO-K1 cell lines by S. Typhimurium was determined by using a standard invasion assay. These data suggested that wild type strains caused invasion to a greater extent than mutant strains. Earlier the authors observed hemagglutination by Salmonella expressing PagN in only rough strains (lacking O antigen of LPS), but adherence and invasion was observed in case of smooth strain SL1344. Thus, they checked for enhanced adherence and invasion of eukaryotic cells by a rough strain. When they compared adherence and invasion of epithelial cells by rough S. Typhimurium strain and rough S. Typhimurium strain lacking PagN, a reduction in cell association was observed as observed in case of smooth strain, however, rough mutant strain displayed nearly five fold decrease in cell association which was greater than the decrease observed in smooth strain. The magnitude of the decrease in cell association observed in case of rough strains lacking pagN was similar to that observed in case of infection of cell lines with S. Typhimurium lacking SP-1 encoded T3SS by mutation in invA. Furthermore, loss of both pagN and invA can be complemented by adding the PagN multicopy plasmid, pPagN2.3.

Anti-V3 monoclonal antibodies display broad neutralizing activities against multiple HIV-1 subtypes

This article discusses the results of neutralization experiments conducted to test human antiV3 monoclonal antibody (mAb) against pesudeoviruses which expressed Env protein of different subtypes.
Catarina E. Hioe1,2*, Terri Wrin3, Michael S. Seaman4, Xuesong Yu5, Blake Wood5, Steve Self5, Constance Williams1, Miroslaw K. Gorny1, Susan Zolla-Pazner1,2
1 Department of Pathology, New York University Langone School of Medicine, New York, New York, United States of America, 2 Veterans Affairs New York Harbor
Healthcare System, New York, New York, United States of America, 3 Monogram Biosciences, Inc., South San Francisco, California, United States of America, 4 Department of Medicine, Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America, 5 Public Health Sciences Division, Statistical Center for HIV/AIDS Research and Prevention, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America

Before starting this article, let me quickly revise the structure of HIV virus (Figure). HIV consists of an outer envelope, from which 72 spikes which are formed of gp 120 and gp 41 are projected. Just below the envelope is present matrix, which is made up of the protein p17. The capsid of the virus is bullet shaped and made up of p24. With in the capsid are present enzymes and two identical strands of RNA.


Figure (Source: http://www.avert.org/aids-picture.php?photo_id=504)

Gp120 has been shown to be a critical target for neutralizing antibodies. It serves a crucial function in the pathogenesis of the virus, i.e. as an attachment protein for attachment of the virus to the host cell. Because gp120 plays such important role in virus pathology, it is logical to think that it as a potential target for neutralizing antibodies. However, gp120 displays high antigenic and genetic variability while its conserved regions are poorly immunogenic and are not assessable on the surface of virion at all the time. The polypeptide sequence of this virus consists of five highly variable regions (V1to V5) which are interspersed with five relatively conserved regions. The first four variable regions form disulphide bonded loops which are thought to be flexible.

In the present study, the authors have evaluated the extent and potency of virus neutralization by mAbs specific for epitopes in the V3 loop. The V3 loop was identified as the target of neutralizing antibodies but it was considered inappropriate as it shows high sequence variability. Its importance as a principle neutralizing domain is controversial. Given the fact that V3 is a part of gp120 that interacts with chemokine receptors, it must have some conserved structural elements and it must be exposed, at least transiently, for binding of the virus to the receptors. These features might explain the results of some studies where, many anti-V3 mAbs were able to recognize and neutralize diverse HIV-1 isolates.

Thus, the author tested seven anti-V3 mAbs that have been previously shown to have potent and cross clade neutralizing activity. They carried out neutralization experiments in two independent labs. For this, 98 pseudo viruses (psVs) expressing Envs of HIV-1 subtypes A. AG, B, C, and D from patients with acute and chronic HIV-1 infections were tested.
Positive neutralization was determined by each mAb/psV pair on the basis on commonly used criteria, the 50% mAb inhibitory concentration Ic50. In addition, a new statistical based criteria was used which was based on
1. the area under the mAb titration curve (AUC)
2. the slope of the titration curve
3. the background neutralization from irrelevant control mAbs
4. the background from a control psV expressing Env from amphotropic murine leukaemia virus (aMLV)

Methods:
Seven human anti-V3 mAbs were selected from more than 50 mAbs generated in the labs of the authors. Human monoclonal antibodies specific for parvovirus B19 or anthrax protective antigen (PA) were used as negative control.

The HIV neutralization assays
Neutralization Assays with U87 target cells
These were performed using U87 target cell lines expressing CD4, CCR5 and CXCR4. This assay was used to test 57 psVs expressing cloned Env gene populations extracted from viruses in patients plasma. For the neutralization assay, the psVs were first treated with 2 or 3 fold serial dilutions of mAbs and then incubated with the U87 cells. After 72 hrs of infection, the levels of the virus were assessed by measuring the luciferase activity.
Negative controls: anti parvo virus mAb and aMLV Env-expressing psV.
Positive controls: psVs expressing cloned Envs of SF162, JR-CSF and NL4.3.

Neutralization Assays with TZM.bl target cells
These were performed using TZM.bl target cell lines expressing CD4, CCR5 and CXCR4.
Neutralizing activities of the anti-V3 mAb against 41 psVs containing cloned Env genes from neutralization sensitive viruses (tier 1), from clade B and clade C isolates from recent infections (tier 2) and viruses from chronic infections (tier 3) were measured.
The procedures were similar to those for neutralization assays with U87 target cells.
Negative controls: MAbs specific for parvovirus B19 or anthrax protective antigen were tested.


Results
 Comparison of AUC and IC50 values for identifying virus neutralizing activities of anti-V3 mAbs in the U87 assay: When the AUC based statistical analysis was performed, it resulted in the detection of nearly twice as much neutralizing activity as did IC50.

 Neutralizing activities of anti-V3 mAbs against multiple HIV-1 subtypes are detected in U87 assay: Of the 57 psVs tested, 21 of the 27 (78%) psVs with subtype B Envs from acute and chronic infection and 8 of the 10 psVs (80%) with subtype C Envs were neutralized by at least one anti-V3 mAb. While, only one out of the 10 subtype D (10%) and 3 of the 10 subtype A (30%) psVs were neutralized by these.


 Neutralizing activities of anti-V3 mAbs against multiple HIV-1 subtypes are also detected in TZM.bl assay: When same mAbs were tested against remaining 41 psVs, which included 10 tier1, 12 tier 2 subtype B, 12 tier 2 subtype C and 7 tier 3, it was observed that higher levels of neutralization were detected in the TZM.bl assay than in U87 assay. When 50% neutralization was used as a cut off, 22 of 41 psVs (54%) were neutralized.

 Combined data from U87 and TZM.bl assays demonstrate broad neutralizing activities of antiV3 mAbs across Envs from different subtypes and from different stages of infection: When the investigators applied AUC statistical approach to the two independent experiments, 56 0f 98 (57%) psVs were found to be neutralized significantly by more than one anti-V3 mAbs. Now what were these 56 psVs? It was found that these 56 pSVs expressed Envs of diverse subtypes including subtypes A, AG, B, C, and D. thus, the ability of many anti-V3 mAbs to neutralize different subtypes of HIV-1 was observed in both the experiments.

 Different antiV3 mAbs display unique patterns of neutralization: It has been seen in previous studies that anti-V3 mAbs derived from African donor infected with HIV-1 non-B subtypes showed different patterns of psV neutralization than those obtained from subtype B infected individuals. It has also been noted that antibodies derived from non-B subtype infected subjects showed broader and more potent neutralization against non-B viruses. In the present study, four non-B derived mAbs were studied. Out of these, two mAbs showed unique neutralization patterns. They showed remarkable neutralization against subtype C psVs. In contrast, they showed sporadic and less potent neutralization against acute, chronic and tier 2 subtype B viruses.

Conclusions:
In the present study, the authors showed that six of the seven anti-V3 mAbs displayed cross clade neutralizing activities. They also observed that 57% of the pseudo viruses tested in this study were effectively neutralized by these mAbs. The authors used old as well as new statistical methods of analysis and observed that this new method has many advantages over the old ones.